Journal: Cancer Reports

Article Title: Retinoic acid receptor γ is a therapeutically targetable driver of growth and survival in prostate cancer

doi: 10.1002/cnr2.1284

Figure Lengend Snippet: Antagonism of RARγ leads to caspase‐independent apoptosis. (A) Serum‐free adapted LNCaP cells were treated with AGN194310 or AGN205728 as indicated, stained with propidium iodide and analyzed by flow cytometry. (B) Agarose gel electrophoresis analysis of DNA from AGN205728‐treated LNCaP cells. (C) Serum‐free adapted LNCaP cells were treated with 10 −6 M AGN194310, AGN205728,or AGN196996 as indicated. Time course of the appearance of DNA‐strand breaks was assessed by TUNEL assay (TACS‐2 TdT Fluorescein in situ apoptosis detection kit). TUNEL‐positive cells were counted for every 500 cells from five fields from each slide, and three different slides were analyzed for each sample. Results are presented as percentage of apoptotic cells (±SD). (D) Serum‐free adapted LNCaP cells were treated with 10 −6 M AGN205728 and the pan‐caspase inhibitor Z‐VAD‐FMK as indicated and assayed as described above. (E) Serum‐free adapted LNCaP cells were treated with 10 −6 M AGN205728, Z‐VAD‐FMK, or etoposide (positive control) as indicated. Cleavage of caspase 3‐, −8 or −9 was assayed using the caspase‐3 substrate DECD‐AFC, the caspase‐8 substrate IEHD‐AFC or the caspase‐9 substrate LEHD‐AFC, respectively. (F) Serum‐free adapted LNCaP cells were treated with docetaxel (10 −10 M), 5‐fluorouracil (5 × 10 −6 M) or etoposide (5 × 10 −6 M) alone or in combination with AGN205728 (10 −7 M) as indicated. Metabolic activity (intracellular ATP concentration) was measured using the Vialight HS High Sensitivity Cell Proliferation/Cytotoxicity assay. Statistical significance is represented as described in Section

Article Snippet: All‐ trans ‐retinoic acid (Cat. # 0695); the pan‐RAR agonist TTNPB (4‐[( E )‐2‐(5,6,7,8‐Tetrahydro‐5,5,8,8‐tetramethyl‐2‐naphthalenyl)‐1‐propenyl]benzoic acid, Cat. # 0761) ; the RARα‐selective agonist AM580 (4‐[(5,6,7,8‐Tetrahydro‐5,5,8,8‐tetramethyl‐2‐naphthalenyl)carboxamido]benzoic acid, Cat. # 0760) ; the PPARγ‐selective agonist ciglitazone (Cat. # 1307) ; the PPARγ‐selective irreversible antagonist GW9662 (Cat. # 1508) ; the microtubule stabilizer docetaxel (Cat. # 4056); the thymidylate synthetase inhibitor 5‐fluorouracil (Cat. # 3257); and the topoisomerase II inhibitor etoposide (Cat. # 1226) were purchased from Tocris Biosciences.

Techniques: Staining, Flow Cytometry, Agarose Gel Electrophoresis, TUNEL Assay, In Situ, Positive Control, Activity Assay, Concentration Assay, Cytotoxicity Assay

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: Binding of AFM11 and CD19/CD3 BiTEs to recombinant antigens and CD3+ cells. (A) Recombinant CD19-Fc was directly immobilized on the Sensor Chip (CM5). AFM11-His was injected (100 nM), and during the dissociation phase the second antigen (CD3) was injected. (B) Jurkat cells or (C) Raji cells were stained with the indicated concentrations of AFM11-His, CD19M13-sm/CD3LCHC21-ktay BiTE, and CD19HD37/CD3TR66 BiTE on ice. Cell-surface bound antibodies were detected by anti-His mAb followed by FITC-conjugated goat anti-mouse IgG. Mean fluorescence intensities determined by flow cytometry were modeled as a sigmoidal dose-response by non-linear regression to calculate KD.

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Binding Assay, Recombinant, Injection, Staining, Fluorescence, Flow Cytometry

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: Summary of the SPR measurements for AFM11-His and the comparator molecules. SPR measurements for binding to recombinant huCD19-Fc and huCD3γϵ; CD19 HD37 /CD3 TR66 BiTE was measured employing the same protocol as described. 21 The tabulated dissociation constant (K D ), association rate constant (k a ), and dissociation rate constant (k d ) were calculated using the global fit and the 1:1 Binding model across 6 concentration curves (0, 12.5, 25, 50, 100 and 200 nM) for AFM11-His and both comparator molecules

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Binding Assay, Recombinant, Concentration Assay

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: (See previous page). Comparison of cytotoxic properties of AFM11-His and the CD19HD37/CD3TR66 BiTE on cell lines. (A) EC50 and (B) specific lysis were determined in cytotoxicity assays with calcein-labeled CD19+ MEC-1 target cells and primary human T cells as effector cells at an E:T ratio of 25:1 with incubation times ranging between 30 min and 300 min. At each time-point the EC50 (calculated by non-linear regression of the data modeled as a sigmoidal curve) and specific lysis for AFM11-His and CD19HD37/CD3TR66 BiTE were plotted as representative experiments. (C) AFM11-His-mediated target cell lysis by CD4+ and CD8+ T cell subsets. 4 and 23 h FACS-based cytotoxicity assays for AFM11-His and CD19HD37/CD3TR66 BiTE were performed with either unfractionated human T cells or enriched human CD4+ or CD8+ T cells as effector cells, and CD19+ NALM-6 cells as target cells at an E:T ratio of 5:1. The plotted values represent the mean and the SD of duplicate measurements. (D, E) Serial killing mediated by AFM11 and CD19HD37/CD3TR66 BiTE was demonstrated by FACS-based cytotoxicity assays. NALM-6 target cells and enriched human T cells were used at the indicated E:T ratios in the presence of increasing concentrations of AFM11-His and CD19HD37/CD3TR66 BiTE. After 23 h incubation, EC50 (D) and percentage of target cell lysis at 10 pM (E) were calculated for each E:T ratio and plotted as representative experiments.

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Comparison, Lysis, Labeling, Incubation

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: AFM11 does not facilitate activation of human T cells in the absence of CD19+ target cells. Dose-responsive induction of CD25 by AFM11-His (A) and CD69 (B) expression on human T cells was assayed in cultures of human PBMC, B cell-depleted PBMC, and enriched T cells after 48 h incubation. Unfractionated PBMC contained 3.8% CD19+ cells. B cell-depleted cultures possessed 0.6% CD19+ cells, and the enriched T cell cultures contained 0.1% CD19+ cells. The kinetics of CD25 (C) and CD69 (D) expression induced by AFM11-His (10 ng/mL) were determined in human PBMC cultures containing 3.5% CD19+ cells and in B cell-depleted PBMC cultures containing < 0.1% CD19+ cells. A through D present representative experiments. (E) No induction of T cell proliferation in the absence of target cells. Unfractionated PBMC, B cell-depleted PBMC, and enriched unstimulated human T cells were cultured in the presence of increasing concentrations of AFM11-His or OKT3 for 4 days, and then pulsed overnight with BrdU. Incorporation of BrdU was assayed by a BrdU ELISA. Control (ctrl.) cells were cultured in the absence of antibodies. Mean absorbance, and SD of triplicates, measured at 450 nm are plotted. The relative fractions of CD19+ cells were 8% in unfractionated human PBMC, 0.8% in B cell-depleted PBMC, and < 0.1% in enriched T cells.

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Activation Assay, Expressing, Incubation, Cell Culture, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: mAbs

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19 + tumor cells

doi: 10.1080/19420862.2015.1029216

Figure Lengend Snippet: Cytokine release by AFM11-His is strictly dependent on the presence of CD19+ target cells. (A) Cytokine release in cultures of PBMC, B cell-depleted PBMC, and enriched human T cells. Unfractionated human PBMC, B cell-depleted PBMC, and enriched T cells were cultured in the presence of: i) 10 µg/mL AFM11-His or OKT3, ii) 100 µg/mL PHA, or iii) without antibodies (w/o). After 48 h incubation IL-2, IL-6, IL-10, TNF, and IFN-γ in the cell-free culture supernatant were quantified by multiplexing and the results of representative experiments were plotted. (B) TNF, (C) IL-2, (D) IL-6, and (E) IFN-γ levels in culture supernatants of PBMC from 7 individual donors. Cytokine levels were determined by Luminex upon stimulation with antibodies in solution. PBMC were either stimulated with AFM11 (open circles) or OKT3 (closed triangles) at the indicated concentrations. IL-2 and TNF levels were measured after 24 h, IL-6 and IFN-γ levels were measured after 48 h. Background (asterisks) was measured in cultures supplemented with vehicle (formulation buffer) corresponding to the volume of highest AFM11 concentration used. Horizontal bars indicate mean values across the 7 donors.

Article Snippet: B cells were depleted from isolated PBMC (EasySepTM Human CD19 Positive Selection Kit, Stem Cell Technologies, Grenoble, France, cat: 18054) according to manufacturer's instructions.

Techniques: Cell Culture, Incubation, Multiplexing, Luminex, Formulation, Concentration Assay