Journal: HemaSphere

Article Title: Combination of targeted pharmacotherapy and immunotherapy with anti‐CD19 CAR NK cells in acute lymphoblastic leukemia

doi: 10.1002/hem3.70238

Figure Lengend Snippet: Efficient transduction of primary human NK cells isolated from eight different donors . (A) Primary human NK cells were isolated and transduced with an EGFP‐encoding alpharetroviral supernatant, and EGFP expression was analyzed using flow cytometry. The expression level on Day 4 is shown as a percentage of EGFP. Shown are the mean values ±SD from n = 8. (B) Dot plots of a representative experiment showing EGFP protein in nontransduced (upper blots) and EGFP‐transduced NK cells (lower blots) isolated from one donor. (C) NK cells were transduced with aCD19 CAR‐expressing alpharetroviral supernatant. Analysis of aCD19 CAR‐transduced NK cells were compared to nontransduced NK cells by flow cytometry. The expression level on Day 4 is shown as a percentage of aCD19 CAR expression. Shown are the mean values ±SD n = 8. ( D ) Dot plots of a representative experiment showing aCD19 CAR surface expression in NK cells (lower blots) or nontransduced NK cells (upper blots) isolated from one donor. (E) The aCD19 CAR‐expressing NK cells were detected by western blot analysis using the CD3ζ antibody. It also detects the endogenously expressed protein CD3ζ, marked with an asterisk (15 kDa). Ponceau S served as a loading control. Lane 1 shows wild‐type NK cells, lane 2 EGFP‐expressing cells, and lane 3 aCD19 CAR‐expressing NK cells. (F) NK cell cytotoxicity against K562 cells was analyzed by coculture of wild‐type or EGFP‐expressing NK cells and K562 (E:T ratio 1:1) for 1–4 h. Data show the mean values ±SD. (G) Degranulation assay of NK cells expressing EGFP. Extracellular CD107a/CD56 expression was analyzed by coculture of wild‐type or EGFP‐expressing NK cells and K562 cells (E:T ratio 1:1, 1 h). The graph represents the mean values ±SD. (H) Cytotoxic activity of wild‐type or aCD19 CAR NK‐expressing NK cells against 697 cells (E:T ratio 1:1). After 4 h of coincubation, the cytotoxicity was determined by flow cytometry. Student's t ‐test was used to compare cytotoxic activity, **** P ≤ 0.0001.

Article Snippet: Using the MACSxpress® Whole Blood NK Cell Isolation Kit and the MACSxpress Erythocyte Depletion Kit (both Miltenyi Biotech), non‐target cells (e.g., T‐, B‐cells, monocytes, and erythrocytes) were removed according to the manufacturer's instructions.

Techniques: Transduction, Isolation, Expressing, Flow Cytometry, Western Blot, Control, Degranulation Assay, Activity Assay

Journal: HemaSphere

Article Title: Combination of targeted pharmacotherapy and immunotherapy with anti‐CD19 CAR NK cells in acute lymphoblastic leukemia

doi: 10.1002/hem3.70238

Figure Lengend Snippet: Cytotoxic activity of primary human NK cells expressing aCD19 CAR against different BCP‐ALL PDX cells . (A) Coincubation experiments of wild‐type and aCD19 CAR NK cells with BCP‐ALL PDX cells ( n = 11) were carried out at various ratios (E:T: 0.1:1, 0.2:1, 0.5:1, and 1:1) for 24 h and the degree of cell lysis was analyzed by flow cytometry. (B) Different transduced aCD19 CAR NK cell donors ( n = 4) were tested for their cell lysis against PDX cells ALL11 at a ratio of 1:1 after 4 and 24 h of coculture. Cytotoxic effects were analyzed by flow cytometry. Data show the mean values ±SD. (A) Student's t ‐test or (B) one‐way ANOVA with the Bonferroni post hoc test were used to compare cytotoxic activity statistically. * P ≤ 0.05), ** P ≤ 0.01, and **** P ≤ 0.0001.

Article Snippet: Using the MACSxpress® Whole Blood NK Cell Isolation Kit and the MACSxpress Erythocyte Depletion Kit (both Miltenyi Biotech), non‐target cells (e.g., T‐, B‐cells, monocytes, and erythrocytes) were removed according to the manufacturer's instructions.

Techniques: Activity Assay, Expressing, Lysis, Flow Cytometry

Journal: HemaSphere

Article Title: Combination of targeted pharmacotherapy and immunotherapy with anti‐CD19 CAR NK cells in acute lymphoblastic leukemia

doi: 10.1002/hem3.70238

Figure Lengend Snippet: aCD19 CAR NK cell therapy dampens leukemia progression, with a mild survival benefit. (A) Treatment scheme of an in vivo PDX experiment with ALL6. (B) ALL6 BCP‐ALL PDX cells were intravenously transplanted in the tail vein of NSG mice. Tumor monitoring was performed by in vivo bioluminescence imaging (BLI) every week. After confirmed engraftment, mice were randomly allocated to the control or treatment group. The treatment group received injections of 1 × 10 7 aCD19 CAR NK cells i.v. over 3 consecutive weeks. During the treatment, mice received 2500 U IL‐15 intraperitoneal thrice weekly to improve NK cell persistence in vivo. (C) BLI signals were quantified by Living Image 4.7.4 software. *** P < 0.001 two‐way ANOVA. (D) Kaplan–Meier survival curve of ALL6 untreated leukemia control mice and aCD19 CAR NK cell‐treated mice. ** P < 0.01 log‐rank test.

Article Snippet: Using the MACSxpress® Whole Blood NK Cell Isolation Kit and the MACSxpress Erythocyte Depletion Kit (both Miltenyi Biotech), non‐target cells (e.g., T‐, B‐cells, monocytes, and erythrocytes) were removed according to the manufacturer's instructions.

Techniques: In Vivo, Imaging, Control, Software

Journal: HemaSphere

Article Title: Combination of targeted pharmacotherapy and immunotherapy with anti‐CD19 CAR NK cells in acute lymphoblastic leukemia

doi: 10.1002/hem3.70238

Figure Lengend Snippet: Enhanced cytotoxicity of a combination of pharmacotherapy and cellular aCD19 CAR NK cell therapy in vitro. BCR‐ABL‐negative ALL (A , B) or BCR‐ABL‐positive ALL (C – F) PDX cells were treated with IC20‐40 of VEN/DEX (VD) (A , B) or VEN/DEX/DAS (VDD) (C – F) combination therapy. After 20 h of pretreatment, the cells were mixed with aCD19 CAR NK cells and incubated for 4 h. Cytotoxicity was assessed using flow cytometric analysis of CD56/calcein AM/PI staining. Statistical significance was determined by one‐way ANOVA with the Bonferroni post hoc test. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. ANOVA, analysis of variance.

Article Snippet: Using the MACSxpress® Whole Blood NK Cell Isolation Kit and the MACSxpress Erythocyte Depletion Kit (both Miltenyi Biotech), non‐target cells (e.g., T‐, B‐cells, monocytes, and erythrocytes) were removed according to the manufacturer's instructions.

Techniques: In Vitro, Incubation, Staining

Journal: HemaSphere

Article Title: Combination of targeted pharmacotherapy and immunotherapy with anti‐CD19 CAR NK cells in acute lymphoblastic leukemia

doi: 10.1002/hem3.70238

Figure Lengend Snippet: Sequential combination of venetoclax‐based pharmacotherapy and aCD19 CAR NK cell immunotherapy induced complete remissions and prolonged survival in different PDX models in vivo. ( A ) Schematic treatment schedule. (B – G) ALL4 cells (B – D) or ALL6 cells (E – G) were systemically engrafted in NSG mice. After confirmed engraftment (by BLI), mice were randomly allocated to an untreated leukemia control group or treated for 4 weeks orally with (B – D) venetoclax (20 mg/kg) and dexamethasone (1 mg/kg) (VD) 5 days a week in case of ALL4 and (E – G) venetoclax (20 mg/kg), dexamethasone (1 mg/kg), and dasatinib (10 mg/kg) (VDD) 5 days a week in case of ALL6. In Week 4 of pharmaco‐treatment, mice were again randomly allocated either to the WT NK cell or the aCD19 CAR NK cell treatment group. Cellular treatment started in Week 4 of pharmacotherapy by an i.v. application of 1 × 10 7 cells once a week for 3 consecutive weeks. During this time, mice received 2500 U IL‐15 i.p. 3 times a week. (B , E) Representative BLI for one mouse per treatment group. (C) Quantification of BLI (total flux) week 10 after TX (F) event‐free survival (EFS) of ALL6 mice as assessed by BLI. (D , G) Kaplan–Meier overall survival (OS) curve of ALL4 (D) and ALL6 (G) mice. (C) * P < 0.05 Students t ‐test. * P < 0.05 and *** P < 0.001 log‐rank test.

Article Snippet: Using the MACSxpress® Whole Blood NK Cell Isolation Kit and the MACSxpress Erythocyte Depletion Kit (both Miltenyi Biotech), non‐target cells (e.g., T‐, B‐cells, monocytes, and erythrocytes) were removed according to the manufacturer's instructions.

Techniques: In Vivo, Control

Journal: HemaSphere

Article Title: Combination of targeted pharmacotherapy and immunotherapy with anti‐CD19 CAR NK cells in acute lymphoblastic leukemia

doi: 10.1002/hem3.70238

Figure Lengend Snippet: Functional analysis of relapses from ALL4 and ALL6 NSG mice treated with pharmacotherapy and cellular immunotherapy. Functional analysis of the relapsed cells concerning (A , B) cell lysis induced by 24 h in vitro treatment with aCD19 CAR NK cells and (C , D) VEN‐mediated MOMP induction after 3 h in comparison to PDX cells isolated from untreated mice (control). The blue area indicates the 95% confidence interval of the control cells.

Article Snippet: Using the MACSxpress® Whole Blood NK Cell Isolation Kit and the MACSxpress Erythocyte Depletion Kit (both Miltenyi Biotech), non‐target cells (e.g., T‐, B‐cells, monocytes, and erythrocytes) were removed according to the manufacturer's instructions.

Techniques: Functional Assay, Lysis, In Vitro, Comparison, Isolation, Control