Supplementary Material and Methods
Antibodies
The Fc engineered CD19 antibody MOR208 (ref. 1), its CD19 IgG1 analogue (containing a
wildtype Fc and identical v-regions) and the control antibody XmAb4614 were kindly
provided by Xencor (Monrovia, CA; USA). Rituximab was obtained from Roche Pharma AG
(Grenzach-Wyhlen, Germany). Antibodies against HLA-A, B, C, CD16 (both FITC-coupled),
and CD69 (PE-conjugated) were obtained from Beckman Coulter (Krefeld, Germany);
antibodies specific for CD56 (PC7-conjugated), CD20 and CD3 (both FITC-coupled) were
obtained from BD Biosciences (Heidelberg, Germany).
Isolation of effector cells, plasma and ALL blasts
Preparation of mononuclear cells (MNC), polymorphonuclear cells (PMN) and plasma from
peripheral blood was performed as described (2-3). NK cells and monocytes were enriched
from healthy donor derived MNC by negative selection using the NK cell isolation kit and the
monocyte isolation kit II (Miltenyi, Bergisch Gladbach, Germany), respectively, and MACS
technology. NK cells from patients were enriched using CD56 microbeads (Miltenyi). Blood
was drawn after receiving the donors’ written informed consents, in accordance with the
Helsinki Declaration.
Flow cytometric analysis
Flow cytometry was performed employing flow cytometers FC 500 or Coulter EPICS XL
(Beckman Coulter) according to standard procedures.
NK cell activation assay
To analyze NK cell activation, un-stimulated NK cells were mixed with ALL target cells, and
incubated with sensitizing antibodies in a volume of 1 ml. After 20 h the expression of CD69
on CD56-positive / CD3-negative NK cells was analyzed by flow cytometry as described (4).
Cytotoxicity assays
ADCC and complement dependent cytotoxicity (CDC) were analyzed in
51
Cr release assays
as described (2-3). 51Cr release from triplicates was measured in counts per minute (cpm). The
percentage of target cell lysis was calculated using the formula: % specific lysis =
[(experimental cpm - basal cpm) / (maximal cpm - basal cpm)] × 100.
Data processing and statistical analyses
Graphical and statistical analyses were performed using GraphPad Prism 4.0 software. Pvalues were calculated using the student's t-test or repeated measures Anova and Bonferroni
post-test when appropriate. The null hypothesis was rejected for p < 0.05.
References
1. Horton HM, Bernett MJ, Pong E, Peipp M, Karki S, Chu SY, et al. Potent in vitro and in
vivo activity of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma and
leukemia. Cancer Res 2008; 68 (19): 8049-57.
2. Peipp M, Lammerts van Bueren JJ, Schneider-Merck T, Bleeker WW, Dechant M, Beyer
T, et al. Antibody fucosylation differentially impacts cytotoxicity mediated by NK and
PMN effector cells. Blood 2008; 112 (6): 2390-9.
3. Repp R, Kellner C, Muskulus A, Staudinger M, Nodehi SM, Glorius P, et al. Combined
Fc-protein- and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances
CD16a binding but does not further enhance NK-cell mediated ADCC. J Immunol
Methods 2011; 373 (1-2): 67-78.
4. Kellner C, Hallack D, Glorius P, Staudinger M, Mohseni Nodehi S, de Weers M, et al.
Fusion proteins between ligands for NKG2D and CD20-directed single-chain variable
fragments sensitize lymphoma cells for natural killer cell-mediated lysis and enhance
antibody-dependent cellular cytotoxicity. Leukemia 2012; 26 (4): 830-4.