Table I.

Effect of alanine substitutions on reactivity with anti-FasL mAbsa and FasL cytotoxicityb

FasL MutantsSurfacecNOK1NOK2NOK3RNOK203Cytotoxicity
Y196Ad
F197A
R198A0.8760.0000.5900.2040.000
G199A0.1470.0350.0000.0970.000
Q200A>1.000>1.0000.000>1.0000.630
S201A>1.000>1.000>1.000>1.0000.909
C202A
N203A0.000>1.0000.000>1.000>1.000
N204A
L205A0.1070.1740.0000.100>1.000
P206A
L207A
S208A>1.000>1.000>1.000>1.000>1.000
H209A
K210A>1.000>1.000>1.000>1.0000.000
V211A
Y212A0.6520.7810.3500.6970.000
M213A>1.000>1.0000.802>1.0000.407
R214A0.793>1.0000.604>1.0000.579
N215A0.758>1.000>1.000>1.0000.000
S216A0.6380.8090.5850.8850.917
K217A>1.000>1.000>1.000>1.0000.880
Y218A0.3790.6130.6020.5580.000
P219A0.7130.968>1.0000.7681.000
Q220A0.0280.0850.0000.0680.000
D221A0.1450.2180.2660.1750.000
L222A0.4390.5080.3110.4110.000
V223A0.817>1.000>1.0000.8420.550
M224A
M225A
E226A>1.000>1.000>1.000>1.000>1.000
G227A0.4060.6860.8850.5890.800
K228A0.2500.8610.0830.2900.000
M229A>1.000>1.0000.5760.721>1.000
M230A0.9640.3700.0000.1280.000
S231A
Y232A
C233A
T234A0.851>1.0000.6060.708>1.000
T235A0.8000.8620.7230.8230.755
G236A
Q237A0.2470.4450.0910.3790.786
M238A0.8400.0000.0800.0000.000
W239A
A240G
  • a Relative binding of NOK1, -2, and -3 and RNOK203 to mutant FasL was evaluated by ELISA. Relative binding was calculated as follows: concentration of native FasL (OD = 0.3)/concentration of mutant FasL (OD = 0.3). Similar results were obtained from three independent experiments.

  • b Relative cytotoxic activity of mutant FasL was tested against hFas/WR19L cells in a 16-h Alamar blue assay. Relative cytotoxicity = ED50 of mutant FasL/ED50 of native FasL. Similar results were obtained from three independent experiments.

  • c The amino acid residues predicted to be exposed on the FasL trimer surface are indicated by circles.

  • d −, Nonproductive.