Journal: Journal of Immunotherapy

Article Title: Preclinical Optimization of a CD20-specific Chimeric Antigen Receptor Vector and Culture Conditions

doi: 10.1097/cji.0000000000000199

Figure Lengend Snippet: FIGURE 1. Modified extracellular spacer domain abrogates FcγR binding and improves in vivo CAR T-cell function. A and B, NSG mice were inoculated IV with Raji-ffLuc tumors followed 2 days later by infusion of 5×106 CD20-targeted CAR+ T cells (TCM-enriched healthy donor T cells transduced with Leu16-28-BBz and restimulated with CD20+ LCL cells), with or without IP injection of 30 mg IV immune globulin (IVIG) per mouse 4 hours before T-cell infusion. Survival is shown in (A), with the curves compared using a log-rank (Mantel-Cox) test. Tumor burden as measured by bioluminescence imaging (mean ± SD of 3 mice per group) is shown in (B). Results are representative of 2 independent experiments. C, CD64 (FcγRI) binding was tested using Jurkat cells transduced with a wild-type IgG1 Fc spacer CAR (iC9-SP163-1F5-IgG1-28-BB-z; “WT spacer”), CAR with spacer replacement of IgG1 hinge-linker by IgG2 hinge linker a.a. sequence (iC9-SP163-1F5-IgG1mut-28-BB-z; “IgG1mut”), and a CAR construct with deletion of the IgG1 hinge-linker (No linker). Transduced cells were identified by anti-CD19 antibody staining to detect the tCD19 transduction marker, and cells were stained with CD64, 3 days after transduction. The CD19+ population was gated, and frequencies of gated cells are shown in the histograms. D, TCM T cells were transduced with Mock, WT spacer, IgG1mut, and No Linker constructs, and cocultured with K562 or K562/CD64 in serum-free medium. To analyze T-cell activation, cells were collected after 24 hours of coculture and stained with CD3, CD19, CD25, and CD69. Frequencies of CD69+ and CD25+cells (gated on CD3+ CD19+ cells) are listed in the quadrants. E,) NSG mice were inoculated IV with Raji-ffLuc tumors followed 2 days later by infusion of 5×106 CD20-targeted CAR+ T cells transduced with the following 1F5-based third generation lentiviral vectors: WT spacer, the same vector but with CH2 domain deleted (CH3 only), or IgG1mut, or mokc-transduced T cells, and tumor burden was measured by bioluminescence over time (n = 3 mice/group). F, Mice were treated as in (E) but also included the No linker CAR and the IgG1mut CAR with N297Q mutation (iC9-SP163-1F5-NQ-28-BB-z; “IgGmut-NQ”). Kaplan-Meier survival curves are shown (n = 5 mice/group) and compared using a log-rank test. Tumor burden curves for this experiment are shown in Figure S2 (Supplemental Digital Content, http://links.lww.com/JIT/A479).

Article Snippet: Raji (Burkitt lymphoma), Jurkat (T-cell lymphoma), Jeko-1 (mantle-cell lymphoma) and K562 (CD20-negative erythroid leukemia) tumor cell lines were purchased from ATCC (Manassas, VA).

Techniques: Modification, Binding Assay, In Vivo, Cell Function Assay, Transduction, Injection, Imaging, Sequencing, Construct, Staining, Marker, Activation Assay, Plasmid Preparation, Mutagenesis

Journal: Translational Cancer Research

Article Title: Long non-coding RNA (LncRNA) CHROMR promotes the expression of the CNNM1 gene by adsorbing hsa-miR-1299 to obtain drug resistance in diffuse large B lymphoma cells

doi: 10.21037/tcr-22-1087

Figure Lengend Snippet: CHROMR’s role in the DLBCL cell line. (A) After CHROMR overexpression, detect CHROMR, miR-1299, and CNNM1 expression changed in SU_DHL_4 cell line by RT-qPCR. (B) After CHROMR overexpression, we detected apoptosis-related genes and CNNM1 expression by Western Blot. (C) In the case of rituximab killing, flow cytometry detected the proportion of cells in the G2 phase of each group. (D) In the case of rituximab killing, the proportion of apoptosis in each group was detected by flow cytometry. (E) After CHROMER overexpression, we detected cell proliferation ability of each group by cck8. *, P<0.05; ***, P<0.001. NC, negative control; DLBCL, diffuse large B-cell lymphoma; RT-qPCR, real time quantitative polymerase chain reaction.

Article Snippet: When necessary, 17 nM rituximab (MCE) was added to the medium to induce cell death.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Negative Control, Real-time Polymerase Chain Reaction

Journal: Translational Cancer Research

Article Title: Long non-coding RNA (LncRNA) CHROMR promotes the expression of the CNNM1 gene by adsorbing hsa-miR-1299 to obtain drug resistance in diffuse large B lymphoma cells

doi: 10.21037/tcr-22-1087

Figure Lengend Snippet: Verification of the CHROMR/miR-1299/CNNM1 pathway through cell function, RT-qPCR, and Western Blot experiments. (A) After transfection of CHROMR-over plasmid and miR-1299 mimic recovery, we detected CHROMR, miR-1299, CNNM1 expression changes in SU_DHL_4 cell line by RT-qPCR. (B) After transfection of CHROMR-over plasmid and miR-1299 mimic recovery, we detected apoptosis-related genes and CNNM1 expression by Western Blot. + indicates that the substance is transfected, − indicates that the substance is not transfected. (C) In the case of rituximab killing, flow cytometry detected the proportion of cells in the G2 phase of each group. (D) In the case of rituximab killing, the proportion of apoptosis in each group was detected by flow cytometry. ***, P<0.001. NC, negative control; RT-qPCR, real time quantitative polymerase chain reaction.

Article Snippet: When necessary, 17 nM rituximab (MCE) was added to the medium to induce cell death.

Techniques: Cell Function Assay, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Negative Control, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: Rituximab conjugation confers γδ2 T cells with CD20 binding capacity. ( a ) A representative histogram of rituximab conjugation is illustrated. DNA linker-1 and linker-2 were conjugated with γδ2 T cells and rituximab, respectively. Linker-1-conjugated γδ2 T cells and linker-2-conjugated rituximab were mixed and ACE1831, rituximab-linked γδ2 T cells, were generated through DNA hybridization. Un-conjugated γδ2 T cells and ACE1831 were stained with R-phycoerythrin-coupled anti-F(ab’)2 antibody to determine the rituximab conjugation efficiency through flow cytometry. Un-conjugated γδ2 T cells (light blue line) represent negative staining, and efficient rituximab conjugation on ACE1831 (dark blue line) is shown. Percent of Max is the highest point of each peak of the overlaid histogram derived from ACE1831 and γδ2 T cells. ( b ) CD20 binding capacity of ACE1831 and γδ2 T cells was determined through flow cytometry analysis. The cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of human CD20-His recombinant protein, and the CD20-bound cell population was identified through staining with Fluorescein-coupled anti-6X His tag antibody. The study was performed in triplicate in five different experiments, and the representative results are shown. Statistical analysis was performed using the t test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Conjugation Assay, Binding Assay, Generated, DNA Hybridization, Staining, Flow Cytometry, Negative Staining, Derivative Assay, Incubation, Recombinant

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: Rituximab conjugation confers γδ2 T cells with superior cytotoxicity against CD20-expressing cancer cells. ( a – c ) ACE1831 and γδ2 T cells were co-incubated with CD20-expressing cancer cells. ( a ) Daudi, ( b ) Raji and ( c ) rituximab-resistant Raji cells at effector to target (E:T) ratios of 1:1, 2:1, 5:1 and 10:1, analyzed via CellTiter-Glo ® luminescent cell viability assay after 4 h of co-incubation. ( d ) CD107a, ( e ) granzyme B and ( f ) IFNγ of ACE1831 in the absence and presence of Raji cells at an E:T ratio of 2:1 after 2 h of incubation were analyzed via flow cytometry. Each group was performed in triplicate from two donor lots, and the representative results are shown. Statistical analysis was performed using the t test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ( g , h ) ACE1831 and γδ2 T cells were co-incubated with ( g ) CD20-negative K562 and ( h ) donor PBMC cells at effector to target (E:T) ratios of 2:1, 5:1 and 10:1 and analyzed using a CellTiter-Glo ® luminescent cell viability assay after 4 h of co-incubation.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Conjugation Assay, Expressing, Incubation, Cell Viability Assay, Flow Cytometry

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: ACE1831 shows superior potency against CD20-expressing cancer cells in vivo. Four doses of intravenously delivered ACE1831 effectively suppressed tumor growth. Tumor-bearing SCID–Beige mice were treated with ACE1831, γδ2 T cells and a Vehicle (serum-free medium) twice per week for two weeks. ( a ) Tumor burden of mice (n = 5 per group) was determined through bioluminescence imaging. ( b ) The bioluminescence intensity of the tumor burden is presented as mean values ± SD. The difference in mean tumor burden between groups was examined using a two-way ANOVA. ***, p < 0.001. ( c ) The survival rate of mice with different treatments was analyzed using the Kaplan–Meier method. **, p < 0.01. ( d ) The body weight of each group of mice is presented as mean value ± SD.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Expressing, In Vivo, Imaging

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: T cell activation and cytotoxicity mediated by the antigen recognition of ACC-linked antibody. ( a ) Jurkat-NFAT-Luc cells were conjugated with different amounts of rituximab using ACC technology and were stained with anti-F(ab’)2 antibody to examine the levels of rituximab conjugated on Jurkat-NFAT-Luc cells. Un-conjugated Jurkat-NFAT-Luc cells (grey line) represent negative staining, and Jurkat-NFAT-Luc cells with low (light blue) and high (dark blue) rituximab conjugation are shown. Percent of Max is the highest point of each peak of the overlaid histogram. ( b ) Jurkat-NFAT-Luc cells conjugated with different amounts of rituximab were co-incubated with different Raji cell numbers (+, 5 × 10 4 ; ++, 2 × 10 5 ; +++, 5 × 10 5 ), and NFAT signaling activation was determined based on NFAT-regulated luciferase activity. Each condition was applied in triplicate in two different experiments, and the representative results are shown. Mean ± SD. Statistical analysis was performed using a t test. **, p < 0.01; ****, p < 0.0001. ( c ) The effector cells were preincubated with or without 1 μg/mL of TCRγδ blocking antibody for 1 h at 37 °C. After 4 h of co-incubation with Raji cells, cytotoxicity against Raji cells was analyzed using a CellTiter-Glo ® luminescent cell viability assay. Each condition was applied in triplicate in two different experiments, and the representative results are shown. Mean ± SD, *, p < 0.05, ***, p < 0.001. ( d ) Illustration delineating the activation of ACE1831 upon encountering CD20-expressing cancer cells.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Activation Assay, Staining, Negative Staining, Conjugation Assay, Incubation, Luciferase, Activity Assay, Blocking Assay, Cell Viability Assay, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Single-cell transcriptomics analysis reveals that the tumor-infiltrating B cells determine the indolent fate of papillary thyroid carcinoma.

doi: 10.1186/s13046-025-03341-7

Figure Lengend Snippet: Fig. 3 TIL-B cells suppress thyrocyte proliferation in both groups but cluster to format the TLSs in indolent PTC. A, B Histogram of the OD450 absorbance in K1 cells that co-cultured with different proportions of TIL-B cells (A) or TIL-B cells culture supernatants (B) from indolent (left) and progressive (right) PTCs. Cell growth of K1 cells was inhibited by TIL-B cells and culture supernatants from both two groups. The cell growth of K1 cells was determined by CCK8 assays. C Multiplex immunofluorescence analysis of the location of B cells and T cells in the tumor (inside the white dotted circle) and adjacent tumor tissues from indolent (I1, I2) and progressive (P) PTCs. Antibodies against CD4, CD8, CD20, and CD21 were used; CD4 and CD8 were marked for T cells, and CD20 and CD21 were marked for B cells. Cell nuclei counterstained with DAPI. TLS, characterized by B cell aggregated and surrounded by T cells, was exclusively present in the tumor region of indolent PTCs. D Histogram of the number of TLSs in the tumor areas of indolent and progressive PTCs. E Histogram of the proportion of CD20+ B cells in tumor region of indolent and progressive PTCs. A two-sided unpaired Wilcoxon test was performed to compare between groups. * indicates p-value < 0.05, ** indicates p-value < 0.01, *** indicates p-value < 0.001. TLSs, tertiary lymphoid structures. Ctr, negative control

Article Snippet: Then, CD20+ B cells were purified from mononuclear cells using a CD20 MicroBeads human kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Cell Culture, Multiplex Assay, Immunofluorescence, Negative Control

Journal: bioRxiv

Article Title: Unbiased discovery of antibody therapies that stimulate macrophage-mediated destruction of B-cell lymphoma

doi: 10.1101/2024.11.13.623229

Figure Lengend Snippet: a , AlphaFold2 predicted structure of WTa2d1xCD38, a bsAb targeting CD47 and CD38. The Fc region of the anti-CD38 arm is highlighted in blue with its respective scFv region in teal, whereas the Fc region of the WTa2d1 arm is highlighted in red with its respective binding domain in orange. The mutations, “knob” (T270Y,T311Y) and “hole” (Y396T, Y437T) are highlighted at the bottom. b , Maldi-TOF analysis of the protein population of the purified bsAb. Based on the theoretical mass of the possible homo and heterodimers, the two main peaks were assigned to WTa2d1xWTa2d1 and WTa2d1xCD38 species. The abundance of WTa2d1xCD38 was calculated as 94.60% based on the peak area. c , Histogram showing binding of the bsAb to wild-type versus CD47 knockout (KO) cell lines highlights the ability of the two antibody arms to bind to their respective antigens. Binding was detected with an Alexa 647-conjugated anti-human IgG secondary antibody. DLD-1 is a colorectal cancer cell line that expresses CD47 but not CD38. d , Binding of WTa2d1xCD38 to the indicated human lymphoma cell lines and macrophages combined from 3 donors indicates the cell-based affinity for the antibody. Data indicate mean ± SD. e , Co-culture assays using primary human macrophages were used to determine the potency of WTa2d1xCD38 across nine different human B-cell lymphoma cell lines, including the indicated wild-type and MHC-I KO variants. Each curve represents an 8-point titration performed in duplicate for each cell line. Fluorescent area was compared at the last imaging time point and normalized based on the maximum value for each cell line. Data depict mean ± SD. f , Comparison of the fluorescent area at the last time point for different B-cell lymphoma cell lines when treated with 10.54 µg/mL of WTa2d1xCD38 or rituximab. Each point represents the mean value for a different cell line performed in duplicate. Student two tailed paired T-test was used to evaluate the difference between the two groups. Lines show median. g , Comparison of the fluorescent area at the last time point from co-culture assays using the indicated lymphoma cell lines treated with either WTa2d1xCD38 or rituximab. Data depict mean ± SD. *p-value<0.0001 for the indicated comparisons by two-way ANOVA with correction for multiple comparisons. h,i , Macrophage phagocytosis of Raji ( h ) and Toledo ( i ) cells when co-cultured for 2 hours in the presence of the WTa2d1xCD38, rituximab, or an anti-CD47 antibody (clone B6H12). All of the values were normalized against the bsAb and the experiment was performed two independent times with three individual co-culture wells per condition. The dotted lines indicate the mean of the negative control condition performed in one experiment with three co-culture wells. Data depict mean ± SD with analysis by two way ANOVA with Tukey’s multiple comparison test. j,k , Co-culture with human macrophages pooled from multiple donors with Raji ( j ) and Toledo ( k ) lymphoma cells as targets. Co-cultures were treated with 1 μg/ml WTa2d1xCD38 and 10 μg/ml antibodies to different Fc gamma receptors as indicated. Curves indicate mean ± SEM.

Article Snippet: Mice were randomized to treatment cohorts and then subjected to intraperitoneal treatment in vivo with vehicle control, 100 µg WTa2d1xCD38, or 100 µg rituximab biosimilar (BioXCell).

Techniques: Binding Assay, Purification, Knock-Out, Co-Culture Assay, Titration, Imaging, Comparison, Two Tailed Test, Cell Culture, Negative Control

Journal: bioRxiv

Article Title: Unbiased discovery of antibody therapies that stimulate macrophage-mediated destruction of B-cell lymphoma

doi: 10.1101/2024.11.13.623229

Figure Lengend Snippet: a , Experimental setup of a xenograft experiment using Raji cells engrafted subcutaneously into the flanks of NSG mice. b , Growth curves of Raji lymphoma tumors in NSG mice treated with the indicated therapies over time (n = 5 mice per treatment cohort). Tumor growth was evaluated by caliper measurements. Mice were treated with control, 100 µg WTa2d1xCD38, or 100 µg rituximab for 14 days. Data depict mean ± SEM. c , Survival analysis of the indicated treatment cohorts. The median survival of Vehicle and Rituximab groups were 38 and 98 days, respectively, whereas the WTa2d1xCD38 was not reached. Log-Rank test was used to evaluate the difference in the probability of survival. Note rituximab curve is minimally nudged for visualization. d , Experimental setup of the xenograft CNS lymphoma model experiment using Raji cells engrafted stereotactically into NSG mice brains. e , Bioluminescence imaging using luciferase reporters of CNS tumors throughout the experiment period (n = 5 mice per treatment cohort). Mice were treated with vehicle control or 200 µg WTa2d1xCD38 three times per week. f , Survival analysis comparing the vehicle control and WTa2d1xCD38 treatment cohorts. Log-Rank test was used to evaluate the difference in the probability of survival between vehicle and treatment conditions.

Article Snippet: Mice were randomized to treatment cohorts and then subjected to intraperitoneal treatment in vivo with vehicle control, 100 µg WTa2d1xCD38, or 100 µg rituximab biosimilar (BioXCell).

Techniques: Control, Imaging, Luciferase

Journal: bioRxiv

Article Title: Unbiased discovery of antibody therapies that stimulate macrophage-mediated destruction of B-cell lymphoma

doi: 10.1101/2024.11.13.623229

Figure Lengend Snippet: Representation of the biological functions performed by the bsAb to enhance macrophage-mediated destruction of B-cell lymphoma cells. The WTa2d1xCD38 bispecific can act as an opsonin and engage Fc receptors on macrophages (1). It can also block immunosuppressive pathways on both the cancer and macrophage cell surface (2 and 3). Additionally, it can enhance the biophysical interactions of macrophages and B-cell lymphoma cells, bringing their cell membranes in close proximity to promote phagocytosis (4). Together, these functions maximally activate macrophage-mediated cytotoxicity of aggressive B-cell lymphoma cells.

Article Snippet: Mice were randomized to treatment cohorts and then subjected to intraperitoneal treatment in vivo with vehicle control, 100 µg WTa2d1xCD38, or 100 µg rituximab biosimilar (BioXCell).

Techniques: Blocking Assay

Journal: Journal of translational medicine

Article Title: SIRPB1 regulates inflammatory factor expression in the glioma microenvironment via SYK: functional and bioinformatics insights.

doi: 10.1186/s12967-024-05149-z

Figure Lengend Snippet: Fig. 4 SIRPB1 Knockout and Macrophage Polarization. A T7E1 assay results. WT wild-type, NC negative control, PC positive control. B SIRPB1 and FLAG-cas9 expression in THP-1 lines. C Sanger sequencing of SIRPB1WT and SIRPB1KO. D Protein expression post-M1/M2 treatments. E mRNA levels of M1/M2 markers (*P < 0.05, **P < 0.01, ***P < 0.001, Dunnett’s test). F Flow cytometry of CD11b, CD86, CD206 in THP-1 lines

Article Snippet: The induced THP-1 macrophages were collected, Fc receptors were blocked by human Fc Receptor Blocking Solution (Maokangbio), stained with FITC Anti-Mouse/ Human CD11b Antibody (Elabscience, clone:M1/70), 7-AAD (Elabscience), APC Anti-Human CD206 Antibody (Elabscience, clone:15–2) and PE Anti-Human CD86 Antibody (Elabscience, clone:BU63), and detected and analyzed by Beckman cytoflex flow cytometry.

Techniques: Knock-Out, Negative Control, Positive Control, Expressing, Sequencing, Flow Cytometry

Journal: British journal of haematology

Article Title: Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL.

doi: 10.1111/bjh.13764

Figure Lengend Snippet: Fig. 1. Obinutuzumab significantly enhanced direct cell death and reduced cell proliferation compared to rituximab in Burkitt Lymphoma (BL) and pre -B-ALL cell lines: (A) Rituximab-sensitive (Raji), Rituximab-resistant (Raji2R and Raji4RH), (B) U698-M -and T-ALL (Loucy) cell lines were treated with 100 lg/ml of obinutuzumab, rituximab (RTX) and isotype control IgG (IgG) for 48 h. Apoptosis was determined by Annexin V/7AAD assay and analysed by flow cytometry. Data shown are mean SD (n = 5, P = 0001, 0001, 0001 and 0001) (C) Raji. Raji2R, Raji4RH U698-M and Nalm-6 cell lines were treated with 100 lg/ml of obinutuzumab, rituximab and isotype control (IgG) for 48 h. Cell proliferation was determined by alamar blue dye and analysed by flow cytometry. Data shown are mean SD (n = 3, P = 00001 and P = 0001). AnxV, Annexin V; PI, propidium iodide; ALL, acute lymphoblastic leukaemia

Article Snippet: Human B-lymphoma Raji (CD20+) and Loucy T-cell acute lymphocytic leukaemia (T-ALL) (CD20- negative control) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) (CCL-86, CRL2629); precursor-CD20+ lymphoblastic leukaemia U698-M (CD20+) and pre-B-ALL, Nalm-6 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) (ACC4,ACC128) and rituximab-resistant cell lines Raji2R and Raji4RH were generously supplied by Matthew J. Barth, MD, and Myron S. Czuczman, MD (Roswell Park Cancer Center, Buffalo, NY, USA).

Techniques: Control, Cytometry

Journal: British journal of haematology

Article Title: Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL.

doi: 10.1111/bjh.13764

Figure Lengend Snippet: Fig. 2. Obinutuzumab significantly enhanced caspase 3/7 activity compared to rituximab in Burkitt Lymphoma (BL) and pre -B-ALL cell lines: (A) BL: Raji, Raji2R, Raji4RH and (B) pre-B-ALL: U698-M cell, Nalm-6 (low CD20 expression) and T-ALL (Loucy) cell lines were treated with 100 lg/ml of obinutuzumab, rituximab and iso- type control IgG for 48 h. Caspase 3/7 activity was determined by pan caspase 3/7 activity by flow cytometry. Data shown are mean SD. (n = 4) (P = 0001, P = 0001, P = 0001 and P = 0001). ALL, acute lymphoblastic leukaemia.

Article Snippet: Human B-lymphoma Raji (CD20+) and Loucy T-cell acute lymphocytic leukaemia (T-ALL) (CD20- negative control) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) (CCL-86, CRL2629); precursor-CD20+ lymphoblastic leukaemia U698-M (CD20+) and pre-B-ALL, Nalm-6 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) (ACC4,ACC128) and rituximab-resistant cell lines Raji2R and Raji4RH were generously supplied by Matthew J. Barth, MD, and Myron S. Czuczman, MD (Roswell Park Cancer Center, Buffalo, NY, USA).

Techniques: Activity Assay, Expressing, Control, Cytometry

Journal: British journal of haematology

Article Title: Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL.

doi: 10.1111/bjh.13764

Figure Lengend Snippet: Fig. 3. Obinutuzumab is significantly more effective in inducing NK cell-mediated ADCC activity compared to rituximab: (A) Raji, Raji2R, Raji4RH and (B) U698M cell lines were treated with obinu- tuzumab, rituximab and IgG isotype control (100 lg/ml) and incu- bated with expanded and activated NK cells for an additional 4 h at 37°C at a 20:1 effector:target ratio. Cell lysis was determined by manufacturer’s protocol using DELFIA cell cytotoxicity assays. Data shown are mean SD (n = 4) (P = 0001, 0001 and 00008). ADCC, antibody-dependent cellular cytotoxicity.

Article Snippet: Human B-lymphoma Raji (CD20+) and Loucy T-cell acute lymphocytic leukaemia (T-ALL) (CD20- negative control) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) (CCL-86, CRL2629); precursor-CD20+ lymphoblastic leukaemia U698-M (CD20+) and pre-B-ALL, Nalm-6 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) (ACC4,ACC128) and rituximab-resistant cell lines Raji2R and Raji4RH were generously supplied by Matthew J. Barth, MD, and Myron S. Czuczman, MD (Roswell Park Cancer Center, Buffalo, NY, USA).

Techniques: Activity Assay, Control, Lysis

Journal: British journal of haematology

Article Title: Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL.

doi: 10.1111/bjh.13764

Figure Lengend Snippet: Fig. 4. Obinutuzumab significantly increased NK cell CD107a expression compared to rituximab: (A) Raji, Raji2R and Raji4RH and (B) U698-M tumour targets were treated with (100 lg/ml) of obinutuzumab, rituximab and isotype (IgG) control and incubated for 16 h with expanded/activated NK cells. Percentage of CD107a expression on CD56+ NK cells was analysed by flow cytometry. Data shown are mean SD of three independent experiments. (P = 005, 001, 0001 and 0005).

Article Snippet: Human B-lymphoma Raji (CD20+) and Loucy T-cell acute lymphocytic leukaemia (T-ALL) (CD20- negative control) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) (CCL-86, CRL2629); precursor-CD20+ lymphoblastic leukaemia U698-M (CD20+) and pre-B-ALL, Nalm-6 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) (ACC4,ACC128) and rituximab-resistant cell lines Raji2R and Raji4RH were generously supplied by Matthew J. Barth, MD, and Myron S. Czuczman, MD (Roswell Park Cancer Center, Buffalo, NY, USA).

Techniques: Expressing, Control, Incubation, Cytometry

Journal: British journal of haematology

Article Title: Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL.

doi: 10.1111/bjh.13764

Figure Lengend Snippet: Fig. 5. Obinutuzumab induced more IFN-c compared to rituximab in expanded and activated NK cells: (A) Burkitt Lymphoma (BL) tumour targets (Raji, Raji2R and Raji4RH) and (B) Pre-B-ALL tumour targets were treated with (100 lg/ml) of obinutuzumab, rituximab and isotype (IgG) control and incubated for 16 h with expanded/activated NK cells. Supernatants were harvested after 16 h. Data shown represent mean SD for three independent experi- ments (P = 003 and 00001). ALL, acute lymphoblastic leukaemia, IFN c, c-interferon.

Article Snippet: Human B-lymphoma Raji (CD20+) and Loucy T-cell acute lymphocytic leukaemia (T-ALL) (CD20- negative control) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) (CCL-86, CRL2629); precursor-CD20+ lymphoblastic leukaemia U698-M (CD20+) and pre-B-ALL, Nalm-6 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) (ACC4,ACC128) and rituximab-resistant cell lines Raji2R and Raji4RH were generously supplied by Matthew J. Barth, MD, and Myron S. Czuczman, MD (Roswell Park Cancer Center, Buffalo, NY, USA).

Techniques: Control, Incubation

Journal: British journal of haematology

Article Title: Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL.

doi: 10.1111/bjh.13764

Figure Lengend Snippet: Fig. 7. Effect of Obinutuzumab versus Rituximab versus Isotype control (IgG) in sensitive BL tumour targets in vivo: NSG mice were injected with (A-B) rituximab sensitive BL Raji Luc+ cells by i.v. were treated once a week till 8 weeks, starting on day 6 (black line). The rituximab sensitive Raji (A) survival and (B) tumour burden were measured once a week till day 80. Kaplan Meier survival curve was generated by GraphPad Prism software and Log rank test post-test (Mantel Cox) was used for the statistical significance (P = 005). PBS, phosphate-buffered saline; BL, Burkitt lymphoma; NSG, non-obese diabetic severe combined immunodeficiency gamma.

Article Snippet: Human B-lymphoma Raji (CD20+) and Loucy T-cell acute lymphocytic leukaemia (T-ALL) (CD20- negative control) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) (CCL-86, CRL2629); precursor-CD20+ lymphoblastic leukaemia U698-M (CD20+) and pre-B-ALL, Nalm-6 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) (ACC4,ACC128) and rituximab-resistant cell lines Raji2R and Raji4RH were generously supplied by Matthew J. Barth, MD, and Myron S. Czuczman, MD (Roswell Park Cancer Center, Buffalo, NY, USA).

Techniques: Control, In Vivo, Injection, Generated, Software, Saline

Journal: British journal of haematology

Article Title: Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL.

doi: 10.1111/bjh.13764

Figure Lengend Snippet: Fig. 8. Effect of Obinutuzumab versus Rituximab versus Isotype control (IgG) in rituximab resistant BL tumour targets in vivo: NSG mice were injected with (A–B) rituximab resistant BL Raji4RH Luc+ cells by i.v. were treated once a week for 8 weeks, starting on day 6 (black line). The rituximabresistant Raji 4RH (A) survival (B) tumour burden were measured once a week until day 80. Kaplan Meier survival curve was generated by GraphPad Prism software and Log rank test post-test (Mantel Cox) was used for the statistical significance (P = 002). PBS, phosphate-buf- fered saline; BL, Burkitt lymphoma; NSG, non-obese diabetic severe combined immunodeficiency gamma.

Article Snippet: Human B-lymphoma Raji (CD20+) and Loucy T-cell acute lymphocytic leukaemia (T-ALL) (CD20- negative control) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) (CCL-86, CRL2629); precursor-CD20+ lymphoblastic leukaemia U698-M (CD20+) and pre-B-ALL, Nalm-6 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) (ACC4,ACC128) and rituximab-resistant cell lines Raji2R and Raji4RH were generously supplied by Matthew J. Barth, MD, and Myron S. Czuczman, MD (Roswell Park Cancer Center, Buffalo, NY, USA).

Techniques: Control, In Vivo, Injection, Generated, Software, Saline