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A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34+ hematopoietic stem cells from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34+ blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.


Single cell killing assay
PBMCs were isolated from buffy coats from healthy donors (n=6) via density gradient centrifugation with Ficoll and banked in liquid nitrogen in heat inactivated FBS with 10% DMSO. After thawing, PBMCs were recovered overnight in complete RPMI medium. T-cells were isolated and cultured in RPMI-1640 medium (Thermo Fisher Scientific) with 10% heatinactivated FBS (Sigma-Aldrich), 1% HEPES (Sigma-Aldrich), IL-2 (60 IU/mL) and IL-15 (10ng/mL) (PeproTech and R&D Systems, respectively) for 7 days. Time-lapse live single-cell imaging was performed similarly as previously described. 3,4 In short, T-cells were stained with 5µM CellTrace yellow and KG1a target cells with 0.5 µM CellTrace far red (both from ThermoFisher Scientific) at 37°C for 20 minutes. Stained T-cells and target cells were seeded onto a silicon-glass microchip containing four separate compartments, each with 2288 microwells with the dimensions 80 x 80 x 300 µm 3 (Supplementary Figure 3). The compartments contained either CD34/CD3 (1000 ng/mL) or RSV/CD3 BiTE (1000 ng/mL) or were left untreated. Imaging of the microchip was performed at 10x magnification using a confocal (Zeiss LSM 880) or widefield (Zeiss Axio Observer 7) microscope, both equipped with environmental chambers kept at 37°C and 5% CO2. T-cell dynamics and target cell killing were monitored for 21 hours, with a time resolution of 5 minutes. Briefly, T-cell-mediated target killing was detected either manually by assessing membrane integrity (e.g., blebbing) or by SYTOX Green Nucleic Acid Stain (25mM, ThermoFisher) fluorescent signal from dead cell in the wells and used to quantify the percentage of cytotoxic T-cells over time. The tracking of individual T-cells dynamic was performed manually in wells with Effector:Target ratio of 1:1 to collect information about time to first contact, contact duration, number of contacts and the percentage of cytotoxic T-cells and of lytic contacts. The percentage of cytotoxic T-cells was assessed after correction for the percentage of spontaneous death in each condition (wells containing only a single target cell that died during the assay).

Human primary AML samples
The blood samples from 14 AML patients were collected at Princess Margaret Cancer Centre (Toronto, Canada). PBMCs were cryopreserved and CD34 + AML blasts used as target cells in killing assays. Purified T-cells from DLI were used as effector cells. A first cohort of six AML patients was selected and cells were co-cultured in an Effector:Target ratio of 3:1 for 72h in the presence of 2.5ug/ml of aCD34 or CD34/CD3 BiTE. A second group of eight AML patients was selected and cells were incubated with RSV/CD3 or CD34/CD3 BiTE at 2.5ug/ml concentration for 72h. Bone marrow samples from three AML patients were collected at Karolinska University Hospital (Huddinge, Sweden). Cells were co-cultured with T-cells (n= 5) isolated from heathy donor buffy coats in an Effector: Target

Human hematopoietic stem cell depletion assay
Unmanipulated peripheral blood stem cell graft (PBSC) samples were collected during preparation of graft for hematopoietic stem cell transplantation (HSCT) and processed as previously described. 5 PBMCs were isolated and banked in liquid nitrogen as described above.
Before usage, PBMC were thawed, rested overnight before HSCs were purified by positive selection of CD34 + cells using the CD34 MicroBead Kit UltraPure (Miltenyi Biotec). T-cells were purified from the same graft using negative selection (Pan T-Cell Isolation Kit; Miltenyi Biotec).
Immediately after purification, T-cells and HSCs were seeded at a 3:1 ratio and culture for 48h   anti-HisTag C D Figure 1 detectable secretory His-tagged protein, soluble BiTE was measured in the supernatant of CART.BiTE cells and total BiTE concentration increased over time at an approximate rate of 10 pg d −1 , corrected for T-cell expansion (Fig. 2h).

BiTEs secreted by CAR-T cells recruit bystander effector activity.
Mechanistically, it remained unclear whether secreted BiTEs solely recruit transduced cells, versus redirecting non-transduced bystander T-cells. To characterize the interaction between secreted BiTE and bystander T cells, we used confocal microscopy to visualize the distribution of EGFR-specific BiTEs on both CAR-T cells and untransduced T cells in culture. Transduced cells were identified by expression of the mCherry fluorescent protein. Biotinylated soluble EGFR detected the anti-EGFR scFv, which could be expressed either as a transmembrane protein in the form of a CAR, or as the unbound arm of a BiTE opposite to its binding site for CD3. Positive control CART-EGFR cells successfully bound free EGFR antigen ( Fig. 3a; left panels). Conversely, negative control T cells transduced with CART-EGFRvIII.BiTE-CD19 did not bind EGFR through either their CAR or secreted BiTE components ( Fig. 3a; center panels). In cultures transduced with CART-EGFRvIII. BiTE-EGFR, we found evidence of EGFR-specific BiTEs, bound in

cells alone (black), and UTD T cells cultured with CART-EGFRvIII.BiTE-CD19 cells (light purple) or CART-EGFRvIII.BiTE-EGFR cells (light orange). UTD T cells stained with concentrated supernatant (~1,000×) from respective cultures are depicted in dark purple and orange, respectively. h, BiTE concentration in supernatant increases over time. UTD T cells (black)
, or those transduced with CART-EGFRvIII.BiTE-EGFR (orange), were cultured with supernatant collected for His-tag ELISA analysis on days 0, 7 and 14. Each experiment was repeated independently at least twice with similar results. Representative data are shown. Assays were performed in triplicate, n = 3 biologically independent wells (mean + s.e.m. is depicted; unpaired, two-tailed t-test, *P < 0.05). Conc, concentrated; VH, variable heavy chain; VL, variable light chain.   . and blotting with anti-His-tag antibody. f, Flow cytometric histograms demonstrating secondary His-tag detection g respective targets. Unconcentrated supernatant from CART-EGFRvIII (green), CART-EGFRvIII.BiTE-CD19 (purple) range) 10 d post-transduction were incubated with K562 cells expressing CD19 or EGFR. g, Flow cytometric g to CD3 on primary human T cells. Data reflect cultures stained with anti-His-tag antibody corresponding to ), and UTD T cells cultured with CART-EGFRvIII.BiTE-CD19 cells (light purple) or CART-EGFRvIII.BiTE-EGFR cells h concentrated supernatant (~1,000×) from respective cultures are depicted in dark purple and orange, respectively. ncreases over time. UTD T cells (black), or those transduced with CART-EGFRvIII.BiTE-EGFR (orange), were cultured ELISA analysis on days 0, 7 and 14. Each experiment was repeated independently at least twice with similar results. s were performed in triplicate, n = 3 biologically independent wells (mean + s.e.m. is depicted; unpaired, two-tailed VH, variable heavy chain; VL, variable light chain.