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T Cells’ Non-Adherent Property is Useful in Cytolytic Assays
(A) A schematic diagram of Cell-Mediated Cytolysis on E-Plate wells. (B)Tumor cells (red) not T cells (blue) elicit impedance. (C) Impedance measurements are transiently disrupted with the addition of T cells to tumor cells. The trace for tumor cells alone is shown in red. The other traces represent tumor cells co-cultured with HER-2/neu p369-specific T cells (Blue: ratio of 1.25 T cells/tumor cell, Green: ratio of 40 T cells/tumor cell).Results are representative of 3 separate experiments. (Data and fi gures adapted from Erskine CL, et. al., 2012).
Killer T Cell Activity is Dose Dependent
(A) Reduction in impedance mediated by T cells is dose dependent. The impedance traces of SKBR3 tumor cells incubated alone or with varying concentrations of tumor-specifi c T cells. Each trace is calculated from triplicate data points collected every 5 minutes through the duration of the culture. Results are representative of 3 separate experiments. (B) The impedance based assay is more sensitive than the chromium release assay for the detection of tumor antigen-specifi c T cells. Shown are % lysis of SKBR3 in response to varying concentrations of p369-specific T cells as measured at 5 hours following tumor and T cell mixing using both the impedance-based assay and the chromium release assay. (Data and figures adapted from Erskine CL, et. al., 2012).
Comparison of Cell Index and [ H]thymidine Incorporation in NK-92-mediated Killing
(A) Representative changes of CI after plating of MCF-7 cells. After 13 h the MCF-7 cells [8,000 alone(black) or plus NK-92 cells at an effector: target cell ratio of 2:1(red), 5:1(green), or 20:1(blue)] were pulsed-labeled with 0.5 µCi of [3H]thymidine (left arrow) for 5 h before NK-92 cells were added. (B) At different times after NK-92 addition, the E-plates were washed with medium, CI was measured, and subsequently the amount of radioactivity was determined. The [3H]thymidine content of MCF-7 cells is shown at different time points [2.5 h (black column), 4.5 h (dark gray column), 6.0 h (light gray column), and 23 h (white column)] at three NK-92 densities (effector:target 0:1, 5:1, and 20:1) (n 4). Statistical analysis (analysis of variance) showed that the decrease of [3H]thymidine content was highly signifi cant (P 0.001) for all time points except 2.5 h. CPM, counts per minute. (Data and fi gures adapted from Glamann J, et. al., 2006).
Detection of Antibody Dependent Cell-mediated Cytolysis (ADCC)
(A) The changes of CI (normalized) before and after inducing ADCC by adding Erbitux and bulk clone NK cells to 16 e-plates devices seeded with A431cells (8,000 cells per well) at time 0. The left arrow marks addition of antibodies, and the right arrow indicates the addition of NK cells (160,000 per well; 20:1 effector:target cell ratio). (B) The concentration–response curve of Erbitux (50% effective concentration 1.3 ng/ml) 10 h after addition of NK cells. (Data and fi gures adapted from Glamann J, et. al., 2006).
Comparison of LAK Cell Killing and Development of NK Cytotoxicty
(A) Lymphokine activated NK (LAK) cell killing of NT2A2 by four different NK donors. The addition of the IL-2 activated NK cells is marked by the vertical bar at 23.30 h. Its shows the profi le of killing across the 80 h time course. (For the generation of LAK-activity , NK cells were cultured in the presence of human recombinant IL-2 (Peprotech) 50 ng/ml for 20 h.) (B) Development of NK cytotoxicity. The infl uence of the target NT2As on the generation LAK-activity was investigated by adding IL-2 (50 ng/ml) to NK-astrocyte co-cultures. (The IL-2 was added to the co-cultures 5 min after the NK cells were seeded onto the target NT2As.) (Data and fi gures adapted from Moodley K, et al., 2011).
Measuring Potency of Bispecifi c Antibody (XGFR) Targeting IGF-1R and EGFR
(A) The schematic diagrams show the one-arm single chain Fab bispecifi c antibody XGFR, and the XGFR2 antibody with C-terminal attachment of disulfi de-stabilized scFvs, and the dual V domain (DVD) antibody XGFR-DVD. All VH and VL domains in the bispecifi c antibodies are derived from the parental antibodies GA201(EGFR; red and yellow) and R1507 (IGF-1R; blue and light blue). (B) Cells were incubated at an effector/tumor cell ratio of 3:1 for 5 h at the indicated concentrations of XGFR, XGFR2, and the parental control antibodies R7072 and GA201 in triplicate in two or more independent experiments. The xCELLigence technology and software was used for data analysis. (Data and fi gures adapted from Schanzer JM, et. al., 2014).
SELECT Publications for Cell-Mediated Cytotoxicity and adcc
1. CAR-T Cells Inflict Sequential Killing of Multiple Tumor Target Cells.
Davenport AJ, Jenkins MR, Cross RS, Yong CS, Prince HM, Ritchie DS, Trapani JA, Kershaw MH, Darcy PK, Neeson PJ.
Cancer Immunol Res. 2015 May;3(5):483-94.
2. Novel bispecifi c antibodies increase γδ T-cell cytotoxicity against pancreatic cancer cells.
Oberg HH, Peipp M, Kellner C, Sebens S, Krause S, Petrick D, Adam-Klages S, Röcken C, Becker T, Vogel I, Weisner D, Freitag-Wolf S, Gramatzki M, Kabelitz D, Wesch D.
Cancer Res. 2014 Mar 1;74(5):1349-60.
3. A Novel Glycoengineered Bispecifi c Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties.
Schanzer JM, Wartha K, Croasdale R, Moser S, Künkele KP, Ries C, Scheuer W, Duerr H, Pompiati S, Pollman J, Stracke J, Lau W, Ries S, Brinkmann U, Klein C, Umana P.
J Biol Chem. 2014 Jul 4;289(27):18693-706.
4. Enzymatic discovery of a HER-2/neu epitope that generates cross-reactive T cells.
Henle AM1, Erskine CL, Benson LM, Clynes R, Knutson KL.
J Immunol. 2013 Jan 1;190(1):479-88.
5. Epigenetic modulation to enable antigen-specifi c T-cell therapy of colorectal cancer.
Chou J, Voong LN, Mortales CL, Towlerton AM, Pollack SM, Chen X, Yee C, Robbins PF, Warren EH.
J Immunother. 2012 Feb-Mar;35(2):131-41.
6. Determining optimal cytotoxic activity of human Her2neu specifi c CD8 T cells by comparing the Cr51 release assay to the xCELLigence system.
Erskine CL, Henle AM, Knutson KL.
J. Vis Exp. 2012;66:e3683.
7. Real-time profi ling of NK cell killing of human astrocytes using xCELLigence technology.
Moodley K, Angel CE, Glass M, Graham E.
J Neurosci Methods. 2011;200(2):173-180.
8. Pertuzumab in combination with trastuzumab shows signifi cantly enhanced antitumor activity in HER2-positive human gastric cancer xenograft models.
Yamashita-Kashima Y, Iijima S, Yorozu K, Furugaki K, Kurasawa M, Ohta M, Fujimoto-Ouchi K.
Clin Cancer Res. 2011;17(15):5060-70.
9. Unique functional status of natural killer cells in metastatic stage IV melanoma patients and its modulation by chemotherapy.
Fregni G, Perier A, Pittari G, Jacobelli S, Sastre X, Gervois N, Allard M, Bercovici N, Avril MF, Caignard A. Clin
Cancer Res. 2011;17(9):2628-37.
10. Dynamic detection of natural killer cell-mediated cytotoxicity and cell adhesion by electrical impedance measurements.
Glamann J and Hansen AJ.,
Assay Drug Dev Technol. 2006;4(5):555-63.
• Real-time monitoring of cell-mediated cytotoxicity, allows measurement of both fast killing kinetics (hrs) and slow killing kinetics (days). Whereas the standard endpoint assays such as the LDH assay and Cr51 release assay can only focus on short term (hrs) cytolysis.
• Label-free, requires less cells compared to the conventional Cr51 release assay, which needs to label target cells with radioactive isotopes.
• Direct, sensitive and specifi c measurement of target cell changes. This is in contrast to the assays based on release of nonradioactive compounds from effector cells.
• Homogeneous assay. This is in comparison to the ATP production assays that require to wash effector cells out of wells first.
• Non-labor intensive assay compared to laborious FACS analysis.