Abstract: Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p & lt; 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p & lt; 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p & lt; 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p & lt; 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p & lt; 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p & lt; 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p & lt; 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p & lt; 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p & lt; 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC & gt; 1 or & lt; -1; p & lt; 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj & lt; 0.0001) and protein secretion (NES=-1.49; p adj & lt; 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.

Abstract: Translation of the success of bispecific T cell engagers (BiTE®) and CAR T cells from B-cell to myeloid malignancies has been challenging. Identifying suitable target antigens in myeloid malignancies has been hampered by expected on-target-off leukemia toxicity. FLT3 is a promising target antigen with high expression in most AML samples independent of FLT3 mutation status (Brauchle et al., 2020). Here, we compared two T-cell based immunotherapy approaches, BiTE® vs CART, in targeting FLT3 in AML. We tested BiTE® and CAR mediated cytotoxicity against several AML cell lines and primary AML (pAML) cells in vitro. The impact of positive co-stimulation was evaluated using our previously established Ba/F3 model system, which is devoid of human costimulatory molecules. Conjugate formation as a surrogate for synapse formation and consecutive T-cell degranulation were analyzed. Combination with tyrosine kinase inhibition (TKI) was tested in conjunction with BiTE® and CAR to further enhance cytotoxicity against FLT3mut AML cells. On-target off-leukemia toxicity was tested in mixing assays of AML cells and healthy bone marrow (hBM). Finally, we compared CAR and BiTE® in an in vivo xenograft AML model. No differences in BiTE® vs CAR mediated cytotoxicity were observed against cell lines with different genetic background and FLT3 expression levels or pAML samples. However, the percentage of T cell-AML conjugates formed utilizing CART was significantly higher compared to BiTE® molecules. Also, faster and stronger degranulation of CD107a was detected using CAR T cells vs BiTE®. Interestingly, we showed that expression of the costimulatory CD86 increased the cytotoxicity of BiTE®-redirected T cells while CAR mediated lysis was unaltered. In case of the FLT3-ITD mutated cell line MV411, the addition of FLT3 TKIs significantly increased CAR and BiTE® mediated cytotoxicity. Looking at on-target off-leukemia toxicity, in particular hBM cells, we only observed low cytotoxicity. Moving to an in vivo AML xenograft model, we observed that CAR T cell treated mice had an improved OS compared to BiTE®-treated mice, accompanied by enhanced proliferation and homing of the CAR T cells to the spleen. We conclude, that FLT3 is a promising target antigen for T cell-based immunotherapy with limited on-target-off leukemia toxicity. Our preclinical in vitro data demonstrate similar cytotoxicity of both platforms against AML cell lines and pAML cells. Utilizing a xenograft mouse model, CAR T cells provided a significant survival benefit over BiTE® molecules. Our data support the hypothesis that positive co-stimulation either integrated within the construct (e.g. 2nd generation CAR) or provided by the target cell (e.g. CD86 expressing leukemia cells) propagates Tcell activation and proliferation. Future studies will be needed to further dissect differences between BiTE® vs CAR T cell based immunotherapy platforms and identify suitable combination partners. Citation Format: Lisa Rohrbacher, Daniel Nixdorf, Helena Stadler, Bettina Brauchle, Florian Märkl, Nora Philipp, Gerulf Hänel, Anetta Marcinek, Maryam Kazerani, Rebecca L. Goldstein, Michael von Bergwelt, Sebastian Kobold, Veit L. Bücklein, Tara Arvedson, Marion Subklewe. The race is on: BiTE vs CART targeting FLT3 in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 894.

Abstract: BiTE ® (Bispecific T-cell Engager) constructs represent a novel immunotherapeutic strategy that recruits T cells against cancer cells independent of their TCR specificity. Currently, two CD33xCD3 BiTE ® antibody constructs (AMG 330 & AMG 673) are being investigated in phase I dose escalation trials in patients with relapsed/refractory Acute Myeloid Leukemia (AML) with early evidence of acceptable safety and anti-leukemic activity (Ravandi et al., ASH 2020; Subklewe et al., EHA 2020). So far, details of BiTE ® mediated T-cell engagement and information on parameters contributing to their efficacy need more investigation. Therefore, we aimed to characterize the interplay between target and effector cells to deepen our mechanistic understanding of BiTE ® construct mediated T-cell engagement. Previously, we have created a novel in vitro model system with murine Ba/F3 cells expressing human (hu) CD33 ± huCD80 ± huCD86 ± huPD-L1 to study T-cell proliferation and cytotoxicity induced by AMG 330. Using that system, we showed that expression of T-cell co-signaling receptors on target cells modulate AMG 330 induced T-cell activity (Marcinek et al., ASH 2018, EHA 2019). Here, we hypothesize that expression of costimulatory molecules impacts BiTE ® mediated immune synapse formation and consecutive downstream signaling in BiTE ® construct activated T cells. To study whether AMG 330 can induce synapse formation and TCR triggering we used a previously described reconstituted T-cell system, which consists of non-immune (HEK) cells introduced with genes encoding the TCR and other proteins (e.g. CD45) required for the regulation of TCR phosphorylation (James et al., Nature 2012). HEK-T cells were incubated with huCD33 transduced RajiB cells in presence of fluorescently labeled AMG 330 or a control BiTE® (cBiTE) construct to allow cell conjugation. A spinning disc confocal microscope system was used to image cells. To pinpoint the role of T-cell co-signaling receptors in immune synapse formation we incubated differentBa/F3 cell constructs or primary AML (pAML) cells with healthy donor T cells in the presence of AMG 330 and analyzed intensity of LFA-1 expression within the synapse using an Imaging Flow Cytometer. Furthermore, we determined phosphorylation of ZAP70, AKT and ERK in conjugated T cells after various time points by phosphoflow cytometry. We observed that AMG 330, in contrast to cBiTE®, induced TCR triggering reflected by exclusion of CD45 from the RajiB-T-cell-interface. Simultaneously clustering of CD33 occurred in AMG 330 induced cell-cell-interfaces (Fig. 1A/B). The percentage of conjugates formed with huCD33 + Ba/F3 cells was significantly higher in constructs expressing huCD86, compared to those expressing no costimulatory antigens or additional huPD-L1 (Mean % in huCD33 + Ba/F3: 2.8 vs. huCD33 + CD86 +.Ba/F3: 4.2 [p=0.0031] vs. huCD33 + huCD86 + PD-L1 + Ba/F3: 2.8 [p=0.0018]). This was accompanied by LFA-1 accumulation within the T-cell-Ba/F3 cell synapse (Mean of MFI in huCD33 + CD86 +.Ba/F3: 10,933 & gt; huCD33 + huCD86 + PD-L1 + Ba/F3: 7,749 & gt; huCD33 + Ba/F3: 7,028). For downstream signaling in T cells after engagement with Ba/F3 cell constructs in the presence of AMG 330, we observed that kinase phosphorylation was highest after 10 minutes in CD86 co-expressing Ba/F3 cells (Mean % of phosphorylation in T-cell conjugates with huCD33 + vs huCD33 + huCD86 + vs huCD33 + CD86 +.PD-L1 + Ba/F3: pERK 40.9 vs 54.3 [p=0.0064] vs 51.2 %; pAKT: 69.1 vs 81.5 [p=0.0642] vs 75.1 %; pZAP70: 6.9 vs 12.2 [p & lt;0.0001] vs 7.7 % [p & lt;0.0001]) (Fig. 1C). Finally, we evaluated if these finding could also be observed in pAML samples. For that, we determined LFA-1 expression intensity within AMG 330-induced pAML-T-cell synapses. We used CD33 + pAML samples with either high CD86 and no PD-L1 expression or vice versa. Comparing synapse formation between these samples, LFA-1 intensity was 4.6-fold higher in the CD86 + PD-L1 - sample compared to the CD86 - PD-L1 + pAML. Taken together, our data unravel molecular mechanisms of BiTE® construct induced immune synapse formation, highlighting the role of costimulatory molecules in this process. They support the notion that T cell co-signaling receptors like CD86 and PD-L1 modulate T-cell response in an early event manner. Prospective analyses in clinical trials are needed to validate the relevance of checkpoint molecule expression on target cells as a potential predictive biomarker for response. Figure 1 Figure 1. Disclosures Brauchle: Adivo: Current Employment. Lacher: Roche: Research Funding. Kischel: Amgen GmbH Munich: Current Employment. von Bergwelt: Roche: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Theurich: Amgen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Buecklein: Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Miltenyi: Research Funding; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Subklewe: Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Klinikum der Universität München: Current Employment; Takeda: 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.

Abstract: Bispecific T-cell engager (BiTE ® ) molecules recruit T cells to cancer cells through CD3ε binding, independently of T-cell receptor (TCR) specificity. Whereas physiological T-cell activation is dependent on signal 1 (TCR engagement) and signal 2 (co-stimulation), BiTE molecule-mediated T-cell activation occurs without additional co-stimulation. As co-stimulatory and inhibitory molecules modulate the strength and nature of T-cell responses, we studied the impact of the expression profile of those molecules on target cells for BiTE molecule-mediated T-cell activation in the context of acute myeloid leukemia (AML). Accordingly, we created a novel in vitro model system using murine Ba/F3 cells transduced with human CD33 ± CD86 ± PD-L1. T-cell fitness was assessed by T-cell function assays in co-cultures and immune synapse formation by applying a CD33 BiTE molecule (AMG 330). Using our cell-based model platform, we found that the expression of positive co-stimulatory molecules on target cells markedly enhanced BiTE molecule-mediated T-cell activation. The initiation and stability of the immune synapse between T cells and target cells were significantly increased through the expression of CD86 on target cells. By contrast, the co-inhibitory molecule PD-L1 impaired the stability of BiTE molecule-induced immune synapses and subsequent T-cell responses. We validated our findings in primary T-cell-AML co-cultures, demonstrating a PD-L1-mediated reduction in redirected T-cell activation. The addition of the immunomodulatory drug (IMiD) lenalidomide to co-cultures led to stabilization of immune synapses and improved subsequent T-cell responses. We conclude that target cells modulate CD33 BiTE molecule-dependent T-cell activation and hence, combinatorial strategies might contribute to enhanced efficacy.

Abstract: Despite advances in the treatment of acute myeloid leukemia (AML), novel therapies are needed to induce deeper and more durable clinical response. Bispecific T-cell Engager (BiTE) molecules, which redirect patient T cells to lyse tumor cells, are a clinically validated modality for hematologic malignancies. Due to broad AML expression and limited normal tissue expression, fms-related tyrosine kinase 3 (FLT3) is proposed to be an optimal BiTE molecule target. Expression profiling of FLT3 was performed in primary AML patient samples and normal hematopoietic cells and nonhematopoietic tissues. Two novel FLT3 BiTE molecules, one with a half-life extending (HLE) Fc moiety and one without, were assessed for T-cell–dependent cellular cytotoxicity (TDCC) of FLT3-positive cell lines in vitro, in vivo, and ex vivo. FLT3 protein was detected on the surface of most primary AML bulk and leukemic stem cells but only a fraction of normal hematopoietic stem and progenitor cells. FLT3 protein detected in nonhematopoietic cells was cytoplasmic. FLT3 BiTE molecules induced TDCC of FLT3-positive cells in vitro, reduced tumor growth and increased survival in AML mouse models in vivo. Both molecules exhibited reproducible pharmacokinetic and pharmacodynamic profiles in cynomolgus monkeys in vivo, including elimination of FLT3-positive cells in blood and bone marrow. In ex vivo cultures of primary AML samples, patient T cells induced TDCC of FLT3-positive target cells. Combination with PD-1 blockade increased BiTE activity. These data support the clinical development of an FLT3 targeting BiTE molecule for the treatment of AML.

Abstract: Bispecific T-cell engaging (BiTE®) antibody constructs recruit T cells to target antigens independent of their T-cell receptor specificity. Blinatumomab, a CD19xCD3 BiTE® antibody construct, is approved for the treatment of relapsed/refractory (r/r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL), including patients with persistence or reoccurrence of measurable residual disease (MRD). Despite superior anti-leukemic efficacy compared to cytostatic agents, a majority of patients do not respond to treatment. Biomarkers for the identification of non-responders prior to or early during treatment are unknown. However, the definition of robust biomarkers for efficacy is of high importance for clinical decision-making and might also indicate ways to improve response rates. We therefore established a comprehensive immune-monitoring program for blinatumomab patients. We sequentially analysed peripheral blood of patients with r/r or MRD-positive disease receiving blinatumomab prior to the start of infusion and once weekly over the course of the first 28-day cycle. We determined CD3+ T cell counts and their subset distribution (CD4, CD8, naïve [TN], central memory [TCM] , effector memory [TEM], and effector memory CD45RA-positive [TEMRA] T cells) by multiparameter flow cytometry (MPFC). Additionally, patient-derived T cells were cocultered with BCP-ALL cell lines (SEM and REH) at an effector: target ratio of 1:3 in presence of blinatumomab or a control BiTE® (0.5 ng/ml, respectively) for 3 days to assess their cytotoxic capacity. Blinatumomab-mediated cytotoxicity was determined by MPFC. T-cell proliferation was evaluated by MPFC (Far Red Cell tracer) after incubation with CD3/CD28 beads for 6 days. A total of 16 patients were enrolled. Four of these patients suffered from MRD disease, whereas the remaining 12 had overt relapse of BCP-ALL. Response rates for patients with morphological relapse were 50% (4 CRMRD- and 2 CRMRD+ after the first cycle) and 75% for patients with MRD disease (3 MRD conversions, 1 MRD persistence). Absolute lymphocyte counts were not significantly different between responders and non-responders before treatment initiation (0.9 G/l and 0.7 G/l, respectively). Whereas the percentage of CD3+ T cells (of all lymphocytes) did not significantly differ between responders and non-responders on day 0, non-responders had a significantly reduced CD3+ percentage on day 7 (81.2% vs 92.9%, p=0.03). Until day 28 of the first cycle, CD3+ percentages of responders and non-responders re-converged (80.2% and 81.4%, respectively). There were no significant differences for CD4+ and CD8+ T-cell percentages prior to and over the course of the first cycle. This was also true for TN, TCM, TEM and TEMRA subset distributions. Additionally, CD19-BiTE®-mediated cytotoxicity of patient-derived T cells was assessed against CD19 expressing target B cell lines in vitro. Interestingly, specific lysis did not differ between responders and non-responders on day 0 (89.6% vs 79.8%, p=0.89), but decreased for non-responders over the course of the first cycle (normalized AUC for cytotoxicity 149 vs 403 in responders, p=0.03). However, this observation was only true for the patients with refractory morphologic relapse, as the non-responding MRD patient maintained T cell cytotoxicity over the first cycle of therapy. Impaired T cell proliferation (leading to reduced E:T ratios) might contribute to the observed dysfunctional cytotoxicity in non-responders, as the percentage of proliferating T cells after CD3/CD28 bead-based stimulation tended to be reduced for non-responders (17.9% vs 36.1%, p=0.07). In summary, lymphocyte counts, T cell percentage and T cell subset distributions do not allow for a response prediction prior to treatment start for patients receiving blinatumomab. BCP-ALL patients with morphological relapse/persistence who do not achieve a remission with blinatumomab therapy show reduced cytotoxicity in vitro in comparison to patients responding to treatment. As this observation could not be confirmed with one MRD non-responder in our cohort, the burden of disease might contribute to the observed T cell dysfunction, possibly by interfering with T cell proliferation. Evaluation of immune checkpoint expression on effector and target cells over the course of the therapy is currently ongoing, as are analyses of the T cell transcriptome of responders and non-responders. Disclosures Kischel: AMGEN Research (Munich) GmbH: Employment, Equity Ownership. Subklewe:Celgene: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Miltenyi: Research Funding; Roche: Consultancy, Research Funding; Janssen: Consultancy; Gilead: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding.

Abstract: Bispecific T-cell engaging (BiTE®) antibody constructs recruit T cells independent of the T-cell receptor specificity. Proof of concept was shown by Blinatumomab, a CD19xCD3 BiTE® antibody construct, for treatment of r/r B-cell precursor ALL. Response rates of 43% led to its approval in 2015, however, the mechanisms of innate and adaptive resistance in non-responders are still incompletely understood. To improve response rates, it is imperative to understand intrinsic and acquired resistance mechanisms. We assessed the relevance of p53 knockdown (kd) in AML cells on cytotoxicity mediated by AMG 330, a CD33 specific BiTE®antibody construct. We initially hypothesized that p53 kd cells would show differential sensitivity to T-cell mediated cytotoxicity. 12 AML cell lines with different p53 status (wt: MV4-11, Molm-13; mut/del: HL60, MONO-MAC 6, K562, Kasumi-1, Kasumi-3, THP-1, SKM-1, NB-4, KG1a) or p53 wt and paired p53 kd cell lines (MV4-11, Molm-13 and OCI-AML3) were cocultured with healthy donor (HD) T cells at an effector: target (E:T) ratio of 1:6 with AMG 330 (5 ng/ml) or a control BiTE®for 3 days. Cytotoxicity was calculated as %specific lysis = 100 × (1 - live CD33+cellsAMG 330/live CD33+cellscBiTE®). To assess T cell proliferation, T cells were stained with the proliferation dye Far Red. For analysis of the costimulatory/coinhibitory expression profile of the paired cell lines, cells were stained for CD80, CD86, CD40L, OX40L, PD-L1, Tim-3 and corresponding isotypes either in steady state or after 72h stimulation with TNFα and IFNγ. To dissect the influence of receptor-ligand interactions versus secretome, we performed transwell experiments. HD T cells and murine Ba/F3 cells (transduced with CD33 and CD86) were cocultured at an E:T ratio of 1:10 in presence of 5 ng/ml AMG 330 or control BiTE®in the lower compartment. In the upper compartment AML cells were cultured, allowing the exchange of soluble factors but no cell-cell contact. Ba/F3 cells were used as a surrogate model to activate T cells and minimize the influence of human checkpoint molecules. To further delineate the influence of p53 kd on T-cell function, we performed transcriptome analysis of the paired AML cell lines. First, 12 AML cell lines were analyzed for AMG 330-mediated cytotoxicity upon coculture with T cells. No difference based on p53 status was observed (p53 wt vs p53 mut (65.86% vs 53.92%; n=27). However, phenotypic and genetic heterogeneity of AML cell lines could mask the influence of p53 mutation on the pharmacodynamic activity of AMG 330. To exclude these cofounding covariables, we performed cytotoxicity assays using p53 wt and paired p53 kd cell lines. AMG 330-mediated cytotoxicity was significantly higher for p53 wt cell lines compared to p53 kd cell lines (wt vs kd: MV4-11: 89.8% vs 72.3%, p 〈 0.03; Molm-13: 90.0% vs 85.2%, p 〈 0.03; OCI-AML3: 89.5% vs 81.2%, p 〈 0.03). Surprisingly, we observed significantly reduced T-cell proliferation in co-cultures with p53 kd cell lines vs p53 wt cell lines (wt vs kd: MV4-11: 74.0% vs 61.6%, p 〈 0.03; Molm-13: 65.7% vs 56.4%, p 〈 0.03; OCI-AML3: 34.9% vs 26.4%, p 〈 0.03). We further demonstrated that the difference is not due to different expression levels of the target antigen CD33 or the costimulatory/coinhibitory molecules CD80, CD86, CD40L, OX40L, PD-L1 and Tim-3 (n= 4). In transwell experiments, T cells proliferated at a lower rate in the presence of p53 kd AML cells in the upper comportment compared to those in the presence of p53 wt AML cells (MV4-11 p53 wt vs p53 kd: 67.9% vs 50.9%, p 〈 0.03) leading to the conclusion that the inhibition of T-cell proliferation is mediated by soluble factors secreted by AML cells. Bulk RNA sequencing of p53 kd AML cell lines compared to p53 wt revealed downregulation of a co-stimulatory ligand 4-1BBL with log2 fold change of - 0.33, -0.30, -0.39 in Molm-13, MV4-11 and OCI-AML3, respectively. In summary, we have observed lower activity of AMG 330 against p53 kd cell lines compared to p53 wt AML cell lines. This correlated with a decrease in T-cell proliferation indicating that the genetic profile of the AML cells modulates T-cell function. We hypothesize that p53 kd AML cells suppress T-cell proliferation, thereby reducing the E:T ratio and redirected cytotoxicity. In a next step, we will try to identify possible secreted mediators of T-cell modulation by mass. We will also characterize primary patient samples for their T-cell-inhibiting capacities, e.g. p53-mutated disease. Disclosures Lichtenegger: Roche: Employment. Kischel:AMGEN Research (Munich) GmbH: Employment, Equity Ownership. Metzeler:Celgene: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Otsuka: Honoraria. Andreeff:NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy. Daver:Celgene: Consultancy; Otsuka: Consultancy; Jazz: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Immunogen: Consultancy, Research Funding; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Agios: Consultancy; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Servier: Research Funding; Incyte: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Glycomimetics: Research Funding; Hanmi Pharm Co., Ltd.: Research Funding; Astellas: Consultancy; NOHLA: Research Funding. Subklewe:Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Morphosys: Research Funding; Janssen: Consultancy; Oxford Biotherapeutics: Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Research Funding; Miltenyi: Research Funding.

Abstract: 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 & lt;0.0001; T-cell expansion as FC: TFI=4±0.8, CONT=1±0.6, p & lt;0.01; Granzyme B MFI ratio of CD8 +: TFI=451±168, CONT=144±33, p & lt;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 & lt;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 & lt;0.0001). DEG analysis of Day 21 TFI vs CONT identified 1417 significantly up- and 1821 downregulated genes (p adj & lt;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.

Abstract: 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.