T-cell-recruiting bispecific molecule therapy has yielded promising results in patients with hematologic malignancies, however, resistance and subsequent relapse remains a major challenge. T-cell exhaustion induced by persistent antigen stimulation or tonic receptor signaling has been reported to compromise outcomes of T-cell based immunotherapies. The impact of continuous exposure to bispecifics on T-cell function, however, remains poorly understood. In relapsed/refractory B-cell precursor acute lymphoblastic leukemia patients, 28-day continuous infusion with the CD19xCD3 bispecific molecule blinatumomab led to declining T-cell function. In an in vitro model system, mimicking 28-day continuous infusion with the half-life-extended CD19xCD3 bispecific AMG 562, we identified hallmark features of exhaustion arising over time. Continuous AMG 562 exposure induced progressive loss of T-cell function (day 7 vs day 28 mean specific lysis: 88.4% vs 8.6%; n = 6; p = .0003). Treatment-free intervals (TFIs), achieved by AMG 562 withdrawal, were identified as powerful strategy for counteracting exhaustion. TFIs induced strong functional reinvigoration of T cells (continuous vs TFI specific lysis on day 14: 34.9% vs 93.4%; n = 6; p < .0001) and transcriptional reprogramming. Furthermore, use of a TFI led to improved T-cell expansion and tumor control in vivo. Our data demonstrate the relevance of T-cell exhaustion in bispecific antibody therapy and highlight that T cells can be functionally and transcriptionally rejuvenated with TFIs. In view of the growing number of bispecific molecules being evaluated in clinical trials, our findings emphasize the need to consider and evaluate TFIs in application schedules to improve clinical outcomes.

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The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

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. Figure 1 Figure 1. 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.