Journal: Cancers

Article Title: Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy

doi: 10.3390/cancers13194785

Figure Lengend Snippet: RevCAR platform targeting CD33 and CD123. ( a ) Schematic overview of the RevCAR system and ( b ) design of the RevTMs. ( a ) RevCARs consist of the extracellular peptide epitope E5B9 or E7B6 and CD28 (28) hinge domain (HiD), CD28 transmembrane domain (TMD), the intracellular CD28 costimulatory (CSD) and CD3 zeta (3z) activating signaling domain (ASD). RevCAR-E5B9-28/3z or RevCAR-E7B6-28/3z modified T-cells are redirected towards CD33 or CD123 expressed on AML blasts via adaptor target modules, named RevTMs. RevTMs are bispecific antibodies (bsAbs) consisting of two different single-chain fragments variable (scFvs) binding on the one hand to E5B9 or E7B6 of the RevCAR and on the other hand to CD33 or CD123 on the surface of AML blasts. ( b ) In detail, the scFvs of the RevTMs are constructed with the variable heavy (V H ) and light chain (V L ) domains derived from the monoclonal antibodies (mAbs) CD33, CD123, 5B9, or 7B6 connected via glycine (G)-serine (S) linkers. RevTMs are expressed in eukaryotic cell lines and secreted into the cell culture supernatant mediated by the Ig kappa leader sequence (Igk). After purification via histidine tag (His), RevTMs were separated by SDS-PAGE and analyzed using Coomassie staining ( c ) and immunodetection after blotting on nitrocellulose membrane via anti-His Ab and AP-conjugated anti-mouse Ab ( d ). The whole western blot figures can be found in .

Article Snippet: After isolation, cells were stained with fluorescently labeled monoclonal Abs (mAbs) directed against human CD3 (#130-113-138), CD33 (#130-113-350), CD45 (#130-092-880) and CD123 (#130-115-265) (all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Modification, Binding Assay, Construct, Derivative Assay, Bioprocessing, Cell Culture, Sequencing, Purification, SDS Page, Staining, Immunodetection, Membrane, Western Blot

Journal: Cancers

Article Title: Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy

doi: 10.3390/cancers13194785

Figure Lengend Snippet: Expression of target antigens and RevCARs and binding capability of RevTMs. ( a ) Number of tumor-associated antigens CD33 or CD123 presented on the surface of MOLM-13, Luciferase (Luc) expressing MOLM-13-Luc or OCI-AML3 cells is shown for three individual experiments as mean ± SD. ( b ) Expression of CD33 and CD123 on MOLM-13 and OCI-AML3 cells was confirmed by flow cytometry after staining with APC-conjugated anti-CD33 mAb or PE-conjugated anti-CD123 mAb. Binding of His tagged RevTMs on AML cells was detected via FITC-conjugated anti-His mAb. ( c ) Number of RevCAR-E5B9-28/3z or RevCAR-E7B6-28/3z on RevCAR modified T-cells is quantified for three independent T-cell donors and represented as mean ± SD. ( d ) Expression of RevCAR-E5B9-28/3z or RevCAR-E5B9-28/3z on modified T-cells was confirmed by staining with anti-E5B9 mAb or anti-E7B6 mAb and PE-conjugated anti-mouse-IgG mAb. Binding of indicated RevTMs to RevCAR-E5B9-28/3z or RevCAR-E5B9-28/3z presenting T-cells was detected using PE-conjugated anti-His mAb. ( b , d ) Stained cells (black lines) and respective controls (grey lines) are presented as histograms. Percentage of positively stained cells is indicated.

Article Snippet: After isolation, cells were stained with fluorescently labeled monoclonal Abs (mAbs) directed against human CD3 (#130-113-138), CD33 (#130-113-350), CD45 (#130-092-880) and CD123 (#130-115-265) (all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Binding Assay, Luciferase, Flow Cytometry, Staining, Modification

Journal: Cancers

Article Title: Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy

doi: 10.3390/cancers13194785

Figure Lengend Snippet: Killing of patient-derived AML blasts by redirected RevCAR T-cells. Expression of ( a ) CD33 and ( c ) CD123 on AML blasts from three patients was assessed by flow cytometry after staining with anti-CD33 and anti-CD123 commercial mAbs. Number of tumor-associated antigens ( a ) CD33 or ( c ) CD123 on the surface of patient-derived AML blasts was determined using QiFi kit. ( b , d ) RevCAR-E5B9-28/3z or RevCAR-E7B6-28/3z T-cells were co-cultured with the eFluor ™ 670-stained patient-derived AML blasts at a ratio of 1:1 in the presence of indicated RevTMs with increasing concentrations in a flow cytometry-based assay, in which the number of living AML blasts was determined. Based on the resulting dose–response curves, half-maximal effective concentration (EC 50 ) values were calculated. Data represents three individual T-cell donors and three individual patient-derived AML blasts as mean ± SD.

Article Snippet: After isolation, cells were stained with fluorescently labeled monoclonal Abs (mAbs) directed against human CD3 (#130-113-138), CD33 (#130-113-350), CD45 (#130-092-880) and CD123 (#130-115-265) (all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Staining, Cell Culture, Concentration Assay

Journal: Cancers

Article Title: Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy

doi: 10.3390/cancers13194785

Figure Lengend Snippet: Combinatorial AND gate targeting of MOLM-13 cells using the Dual-RevCAR system. ( a ) Schematic overview of the Dual-RevCAR system. Dual-RevCAR T-cells express both the activating signaling SIG RevCAR-E7B6-3z and the costimulatory COS RevCAR-E5B9-28. For complete redirection and activation of Dual-RevCAR-E7B6-3z-E5B9-28 T-cells against AML cells, both RevCARs have to be simultaneously engaged via RevTM CD123-7B6 and RevTM CD33-5B9 recognizing CD123 or CD33, respectively. ( b ) Amount of COS RevCAR-E5B9-28 and SIG RevCAR-E7B6-3z receptors on Dual-RevCAR-E7B6-3z-E5B9-28 T-cells. Three individual T-cell donors are represented as mean ± SD. ( c , d ) Proof-of-concept of AND logic gate using the RevCAR system. Dual-RevCAR-E7B6-3z-E5B9-28 T-cells were co-cultured with MOLM-13-Luc cells at indicated e:t ratios in the presence of either the signaling RevTM CD123-7B6, the costimulatory RevTM CD33-5B9 or the combination of both RevTMs. ( c ) In a Luc-based cytotoxicity assay, specific lysis of MOLM-13-Luc cells was calculated for three individual T-cell donors as mean ± SD (Two-way ANOVA with Tukey’s multiple comparisons test. Significance versus w/o RevTM, RevTM CD123-7B6 or RevTM CD33-5B9 alone.). ( d ) Release of the indicated cytokines from redirected Dual-RevCAR-E7B6-3z-E5B9-28 T-cells into the cell culture supernatants of co-cultures was analyzed for three individual T-cell donors as mean ± SD (One-way ANOVA with Tukey’s multiple comparisons test. Significance versus w/o RevTM, RevTM CD123-7B6 or RevTM CD33-5B9 alone). p values below 0.0332 were considered significant: p < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***).

Article Snippet: After isolation, cells were stained with fluorescently labeled monoclonal Abs (mAbs) directed against human CD3 (#130-113-138), CD33 (#130-113-350), CD45 (#130-092-880) and CD123 (#130-115-265) (all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Cell Culture, Cytotoxicity Assay, Lysis

Journal: Cancers

Article Title: Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy

doi: 10.3390/cancers13194785

Figure Lengend Snippet: Combinatorial AND gate targeting of patient-derived AML blasts using the Dual-RevCAR system. ( a ) Expression of CD33, CD123 and CD33 + /CD123 + on patient-derived AML blasts was assessed by flow cytometry after staining with anti-CD33 and anti-CD123 commercial mAbs. ( b ) Number of tumor-associated antigens CD33 or CD123 on the surface of patient-derived AML blasts was determined using QiFi kit. ( c ) AND gate targeting was assessed on patient-derived AML blasts. Dual-RevCAR-E7B6-3z-E5B9-28 T-cells were co-cultured with the eFluor ™ 670-stained patient-derived AML blasts at a ratio of 1:1 in the presence of indicated RevTMs in a flow cytometry-based assay. The SIG RevCAR-E7B6-3z and the COS RevCAR-E5B9-28 receptors on Dual-RevCAR-E7B6-3z-E5B9-28 T-cells were triggered via the RevTM CD33-7B6 and RevTM CD123-5B9, respectively. Data for one sample of patient-derived AML blasts as mean ± SD is shown.

Article Snippet: After isolation, cells were stained with fluorescently labeled monoclonal Abs (mAbs) directed against human CD3 (#130-113-138), CD33 (#130-113-350), CD45 (#130-092-880) and CD123 (#130-115-265) (all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Staining, Cell Culture

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

doi: 10.1186/s13046-021-02093-4

Figure Lengend Snippet: SAMHD1 determines CNDAC sensitivity of primary AML cells. ( A ) Correlation of SAMHD1 protein levels and CNDAC concentrations that reduce cell viability by 50% (IC 50 s) in bone-marrow-derived leukaemic blasts derived from 24 therapy-naïve AML patients. Cells were co-immunostained for CD33, CD34, CD45 (surface markers) and intracellular SAMHD1 and the mean fluorescence intensity (MFI) was analysed by flow cytometry. ATP assays were performed in three technical replicates to determine the CNDAC IC 50 values. Linear regression analyses were performed using GraphPad Prism. ( B ) CNDAC IC 50 values in bone-marrow-derived leukaemic blasts derived from six therapy-naïve AML patients either treated with VPX virus-like particles (VPX-VLPs, cause SAMHD1 depletion), VPR virus-like particles (VPR-VLPs, negative control), or left untreated. Horizontal lines and error bars indicate means ± SD of three technical replicates. p-values were determined by two-tailed Student’s t-tests (* p < 0.05; ** p < 0.01; *** p < 0.001). ( C ) CNDAC dose-response curves in primary AML cells of one exemplary patient (Patient E) treated with VPX-VLPs, VPR-VLPs or left untreated. IC 50 values represent means ± SD of three technical replicates. ( D ) Representative Western blots indicating SAMHD1 levels in primary AML cells derived from Patient E in response to treatment with VPX-VLPs. ( E ) CNDAC-triphosphate (CNDAC-TP) levels as determined by LC-MS/MS in primary AML cells derived from Patient E in response to treatment with VPX-VLPs

Article Snippet: Staining for surface markers (CD33, CD34, CD45) for AML blasts was applied before fixation with the following fluorochrome-conjugated antibodies: CD33-PE and CD34-FITC, both from Miltenyi Biotech (130–111-019, 130–113-178) and CD45-V450 from BD Pharmingen (Heidelberg, Germany, 642,275), all diluted 1:5 per 1 × 10 7 cells, and goat anti-rabbit Alexa-Fluor-660 from Invitrogen, Life technologies (1:200, A-21073) as secondary antibody for SAMHD1 staining.

Techniques: Derivative Assay, Fluorescence, Flow Cytometry, Virus, Negative Control, Two Tailed Test, Western Blot, Liquid Chromatography with Mass Spectroscopy