Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Novel BAFF-receptor antibody to natively folded recombinant protein eliminates drug resistant human B-cell malignancies in vivo

doi: 10.1158/1078-0432.CCR-17-1193

Figure Lengend Snippet: (A) Treatment schedule following Day 0 tumor challenge with minimum lethal dose of tumors. Antibody treatments were given by IV tail vein injections: 200 μg treatment antibody, 10 × 106 effector human NK-92-176V cells, and 5 × 104 IU IL-2. Bioluminescence imaging monitored mice challenged with luciferase-expressing tumors: (B) JeKo-1 (MCL) or (C) RS4;11 (ALL). Experimental groups received treatment of chimeric BAFF-R mAbs (C55 or C90, as indicated). Control group mice received PBS, NK cells alone, or rituximab on the same schedule. Data are representative of three independent experiments.

Article Snippet: Human NK-92 176V cells were obtained from Conkwest Inc. (San Diego, CA).

Techniques: Imaging, Luciferase, Expressing

Journal: Transplantation and cellular therapy

Article Title: NK Cell Adoptive Immunotherapy of Cancer: Evaluating Recognition Strategies and Overcoming Limitations

doi: 10.1016/j.bbmt.2020.09.030

Figure Lengend Snippet: Sample List of Active Trials Involving Allogeneic NK Cells

Article Snippet: Anderson Cancer Center; National Cancer Institute CB-derived NK cells Rituximab Stem cell transplant Recurrent or refractory B cell non-Hodgkin lymphoma NCT03027128 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Metastatic of locally advanced solid tumors NCT03068819 Washington University School of Medicine Cytokine-induced memory-like NK cells CD3 + T cells donor lymphocyte infusion Relapsed AML NCT03136406 NantKwest, Inc. NK92 cell line ALT-803 Other immune modulators Pancreatic cancer NCT03167164 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Merkel cell carcinoma NCT03167177 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Melanoma NCT03169738 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Non-small-cell lung cancer NCT03169764 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Head and neck squamous cell carcinoma NCT03169777 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Colorectal cancer NCT03169790 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Non-Hodgkin lymphoma NCT03175666 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Triple-negative breast cancer NCT03197571 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Urothelial carcinoma NCT03197584 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Ovarian cancer NCT03300492 University Hospital, Basel, Switzerland Haploidentical NK cells Acute myeloid leukemia; myelodysplastic syndromes NCT03329248 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Pancreatic cancer NCT03383978 Johann Wolfgang Goethe University Hospital NK92 cell line gene modified to express anti-Her2 CAR-expressing CD28 and ζ Glioblastoma NCT03387085 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Triple-negative breast cancer NCT03387098 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Pancreatic cancer NCT03387111 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Squamous cell carcinoma NCT03415100 The Third Affiliated Hospital of Guangzhou Medical University Autologous or allogeneic NK cells transfected with mRNA expressing NKG2D-directed CAR Metastatic solid tumors NCT03420963 M.D.

Techniques: Transplantation Assay, Modification, Transfection, Expressing, Derivative Assay

Journal: Oncotarget

Article Title: Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

doi: 10.18632/oncotarget.11019

Figure Lengend Snippet: A. Schematic representation of the CD3CAR lentiviral vector (top). The CD3CAR NK-92 construct is a tandem signaling domain that contains: a leader sequence; an anti-CD3scFv; a hinge domain (H); a transmembrane domain (TM); two co-stimulatory domains (CD28 and 4-1BB) that define the construct as a “third generation” CAR 7 ; and a CD3zeta intracellular signaling domain. B. Western blot analysis of CD3CAR (bottom left). HEK-293FT cells were transduced with lentiviral plasmids for GFP (lane 1) and CD3CAR NK-92 (lane 2) for Western blot analysis at 48h post transduction and probed with mouse anti-human CD3zeta antibody. The expected weight of the CD3CAR NK-92 construct is 58.2 kDa by sequence analysis data (not shown). C. Flow cytometry analysis of CD3CAR NK-92 expression on NK-92 cell surface for vector control NK-92 cells and CD3CAR NK-92 cells (bottom right). Population in green delineates the transduced CD3CAR NK-92 cells. Gating done against isotype controls.

Article Snippet: After 24 hours of co-culture, CD3CAR NK-92 cells efficiently lysed 85% of CCRF-CEM CD3+ cells at an E:T ratio of 2:1, with close to 100% lysis at an E:T ratio of 5:1 (Figure ), suggesting that CD3CAR NK-92 cells successfully ablated even CD3 dim CCRF-CEM CD3+ cells.

Techniques: Plasmid Preparation, Construct, Sequencing, Western Blot, Transduction, Flow Cytometry, Expressing, Control

Journal: Oncotarget

Article Title: Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

doi: 10.18632/oncotarget.11019

Figure Lengend Snippet: A. Co-cultures with CD3CAR NK-92 performed at an effector:target (E:T) ratio of 2:1. T-lymphoblast cell line Jurkat (90% CD3 + ) co-cultured for 6 hours. Sorted CD3 + CCRF-CEM (CCRF-CEM CD3+ ) co-cultured for 24 hours. Negative control, CD3 - non-Hodgkin's Lymphoma cell line KARPAS 299 co-cultured for 24 hours. Target populations quantified with flow cytometry using CD56 and CD3 to distinguish NK and target cell populations. CD4 used to determine KARPAS population. Populations encircled to highlight lysis amount. B. Co-cultures with CD3CAR NK-92 performed at an effector:target (E:T) ratio of 5:1. Experimental conditions identical as 2:1. C. Graphical summary of CD3CAR NK-92 in vitro assays against T-ALL cell lines. Each bar represents the average % cell lysis for duplicate samples; N = 4 experiments for Jurkat and CCRF-CEM CD3+ and N = 2 experiments for KARPAS. D. Absolute cell counts of CCRF-CEM CD3+ and Jurkat cultures with CD3CAR NK-92 cells at an E:T ratios of 5:1. Control and CD3CAR treatment samples are labeled in red and blue respectively with effector and target cell counts performed via FACS analysis from Kaluza flow cytometry software (Beckman Coulter).

Article Snippet: After 24 hours of co-culture, CD3CAR NK-92 cells efficiently lysed 85% of CCRF-CEM CD3+ cells at an E:T ratio of 2:1, with close to 100% lysis at an E:T ratio of 5:1 (Figure ), suggesting that CD3CAR NK-92 cells successfully ablated even CD3 dim CCRF-CEM CD3+ cells.

Techniques: Cell Culture, Negative Control, Flow Cytometry, Lysis, In Vitro, Control, Labeling, Software

Journal: Oncotarget

Article Title: Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

doi: 10.18632/oncotarget.11019

Figure Lengend Snippet: A. Co-cultures were carried out at an E:T ratio of 2:1 for 24 hours. Ability of CD3CAR NK-92 cells to lyse target cells, % cell lysis, determined by relative amounts of residual target cells post co-culture. CD3CAR NK-92 cells lyse patient sample PT4 ( N = 4) (unclassified PTCL phenotype CD7 - CD3 + ) and SPT-1 (Sézary Syndrome) ( N = 2) leukemic cells obtained from patient bone marrow aspirate expressing CD3 as well as normal human T cells (UCB T) isolated from cord blood ( N = 4). B. Co-cultures carried out for 24 hours at an E:T ratio of 5:1. Experimental conditions identical to above. C. CD3CAR NK-92 cells co-cultured with T-ALL patient sample expressing majority CD3 - tumor burden with a small population of normal T-cells (purple). Co-cultures were conducted in E:T ratios of 2:1 and 5:1 for 24 hours. Cytotracker dye (CMTMR) was used to stain T-ALL sample due to heterogeneity in flow phenotype. D. Bar graphs summarizing the cytotoxic activity of CD3CAR NK-92 cells against all types of CD3+ cell populations. T-lymphoblast cell lines and patient samples from T-cell lymphomas expressing CD3 co-cultured with CD3CAR NK-92 cells in the indicated E:T (effector:target) cell ratios of 2:1 and 5:1. % cell lysis values determined using the CD3 + total population as the target population with the exception of PT4, where the CD3 + CD7 - population was designated as the target cell population for enhanced specificity. E. Absolute cell counts of selected PT4 and SPT-1 co-cultures at an E:T ratio of 5:1 over 24 hours. Control and CD3CAR treatment samples are labeled in red and blue respectively. Effector and target cell counts were obtained from Kaluza flow cytometry software (Beckman Coulter).

Article Snippet: After 24 hours of co-culture, CD3CAR NK-92 cells efficiently lysed 85% of CCRF-CEM CD3+ cells at an E:T ratio of 2:1, with close to 100% lysis at an E:T ratio of 5:1 (Figure ), suggesting that CD3CAR NK-92 cells successfully ablated even CD3 dim CCRF-CEM CD3+ cells.

Techniques: Lysis, Co-Culture Assay, Expressing, Isolation, Cell Culture, Staining, Activity Assay, Control, Labeling, Flow Cytometry, Software

Journal: Oncotarget

Article Title: Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

doi: 10.18632/oncotarget.11019

Figure Lengend Snippet: A. Flow cytometry schemes showing dose-dependent CD3CAR NK-92 co-cultures with CCRF-CEM CD3+ and peripheral blood derived (PB) T-cells. E:T ratios were decreased to a lower bound of 0.25:1 (25,000 effector cells to 100,000 target cells) and encircled populations represent lysis effects of interest. Co-cultures were conducted over 24 hours. B. Dose-dependent flow analysis of CD3CAR NK-92 cells with Jurkat cell line. Co-cultures were conducted for 6 hours and populations analyzed. C. Dose-dependent co-cultures of CD3CAR NK-92 cells with PT4 (unclassified PTCL) primary patient samples obtained from bone-marrow aspirate. D. Bar graphs summarizing the dosage relationships between CD3CAR NK-92 dose and target cell depletion. Bars represent the average of duplicate samples with CCRF-CEM CD3+ ( N = 4), Jurkat ( N = 4), PB T-cell ( N = 2), and PT4 ( N = 4).

Article Snippet: After 24 hours of co-culture, CD3CAR NK-92 cells efficiently lysed 85% of CCRF-CEM CD3+ cells at an E:T ratio of 2:1, with close to 100% lysis at an E:T ratio of 5:1 (Figure ), suggesting that CD3CAR NK-92 cells successfully ablated even CD3 dim CCRF-CEM CD3+ cells.

Techniques: Flow Cytometry, Derivative Assay, Lysis

Journal: Oncotarget

Article Title: Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

doi: 10.18632/oncotarget.11019

Figure Lengend Snippet: A. Elimination of luciferase-expressing Jurkat cells in xenografted mice treated with CD3CAR NK-92 as measured via IVIS imaging. NSG mice were sublethally irradiated and, after 24 hours, intravenously injected with 1 × 10 6 luciferase-expressing Jurkat cells (Day 0) to induce measurable tumor formation. Three days (Day 3) following Jurkat cell injection, mice were intravenously injected via tail vein with one course consisting of a total of 15 × 10 6 CD3CAR NK-92 cells or vector control NK-92 cells ( N = 6 per group) during the window of the NK cell life expectancy ending on Day 10. On Day 10, t­­­­wo mice died most likely as a result of stroke from injection procedure and NK cell aggregation. Two additional low dose injections totaling 5 × 10 6 CD3CAR NK-92 cells were administered through Day 14 and 23 to see if this tumor control could be maintained. On days 4, 7, 9, and 13, mice were injected subcutaneously with RediJect D-Luciferin and subjected to IVIS imaging. The % cell lysis by CD3CAR NK-92 relative to control was determined via luciferin signal. B. CD3CAR NK-92 controls Jurkat tumor growth in vivo . Average light intensity (in photons per second) measured for the CD3CAR NK-92 injected mice was compared to that of vector control NK-92 injected mice. P-values are indicated at specific time-points, demonstrating a statistically significant reduction in the relative tumor burden by CD3CAR NK-92 cells as compared to vector control. A zoomed in version of the graph from Days 4-9 is included to better illustrate the error measurements and tumor growth. C. CD3CAR NK-92 reduces Jurkat tumor burden in vivo . Percent luciferin signal measured for CD3CAR NK-92 injected mice and demonstrated as percent difference in signal from vector control NK-92 injected mice. The percent reduction in the tumor burden calculated on days 4, 7, 9, 13, and 17. D. CD3CAR NK-92 treated mice survive significantly longer than control. Kaplan-Meier survival curve for CD3CAR NK-92 treated mice compared to vector control treated mice. Log-rank (Mantel-Cox) test p-values as shown and on Day 43, all CD3CAR-treated mice sacrificed for persistency studies. Two mice that died from the injection procedure were excluded from the survival curve and statistics calculation.

Article Snippet: After 24 hours of co-culture, CD3CAR NK-92 cells efficiently lysed 85% of CCRF-CEM CD3+ cells at an E:T ratio of 2:1, with close to 100% lysis at an E:T ratio of 5:1 (Figure ), suggesting that CD3CAR NK-92 cells successfully ablated even CD3 dim CCRF-CEM CD3+ cells.

Techniques: Luciferase, Expressing, Imaging, Irradiation, Injection, Plasmid Preparation, Control, Lysis, In Vivo