SUPPLEMENTAL TABLES AND FIGURES
Table S1. Affinities relative to hu3F8-IgG1*
Affinity for CD16
Affinity for CD32
FcR
n
Relative to
hu3F8-IgG1
on
CD16A(158F)
CD16A(158V)
3
3
0.2
hu3F8-IgG1 CD16A(158F)
3
1
0.1
Antibody
hu3F8IgG1n
hu3F8-IgG1DEL
hu3F8IgG1n-DEL
n
Relative to
hu3F8-IgG1
on
CD32A(131R)
SD
CD32A(131R)
3
1.0
0.2
CD32A(131H)
3
2.4
0.5
SD
p value
FcR
0.001
p value
A:I
Ratio**
0.02
17
CD16B
3
1
0.6
CD32B
3
0.2
0.1
CD16A(158V)
4
12
2.4
0.004
CD32A(131R)
4
0.3
0.1
0.01
CD16A(158F)
4
5
0.9
0.005
CD32A(131H)
4
0.4
0.4
0.004
CD16B
4
2
0.5
0.07
CD32B
4
0.1
0.1
NS
CD16A(158V)
3
28
9
0.04
CD32A(131R)
3
2.0
1.0
NS
CD16A(158F)
3
18
5
0.03
CD32A(131H)
3
2.5
0.8
NS
CD16B
3
16
4
0.01
CD32B
3
1.9
1.1
NS
CD16A(158V)
6
71
23
0.001
CD32A(131R)
4
0.1
0.1
0.004
CD16A(158F)
6
41
13
0.001
CD32A(131H)
4
0.6
0.9
0.02
CD16B
6
16
5
0.001
CD32B
4
0.1
0.0
NS
106
15
512
* Affinity reference highlighted in grey. All results were fitted with Two-State Reaction:
A+B=AB=AB*, KD = (kd1/ka1)/(1+ka2/kd2).
** A:I ratio = Activating : inhibitory = (relative KA on CD16-158V)/(relative KA on
CD32B).
1
Table S2. Affinities to both human and mouse FcR(n)*
Affinity for human FcR(n)
Affinity for mouse FcR(n)
Relative to
Antibody
KD (nM)
Relative to
KD (nM)
hu3F8-IgG1
hu3F8-IgG1
hu3F8-IgG1
135 ± 24
1.00
61.3 ± 14.0
1.00
hu3F8-IgG1n
119 ± 16
1.13
56.6 ± 13.0
1.08
hu3F8-IgG1-DEL
144 ± 21
0.94
75.6 ± 8.9
0.81
hu3F8-IgG1n-DEL
128 ± 16
1.06
50.7 ± 8.3
1.21
* All results were fitted with steady-state (SS) equilibrium analysis. Fitted data ±
standard error were presented. No significant statistical difference between all four
antibodies. The data were shown as the overall apparent affinity KD, which reflected the
avidity effects due to the bivalency of the FcRn:IgG interaction when FcRn was
immobilized on the sensor chip.
2
A
B
RU
400
350
KD = 158 nM
700
300
600
250
500
Response
Response
RU
800
KD = 837 nM
200
150
100
50
400
300
200
100
0
0
-50
-100
-50
0
50
Time
100
150
200
-100
-100
250
s
C
-50
0
50
Time
100
150
200
250
s
D
RU
1600
RU
KD = 76.4 nM
1400
KD = 27.2 nM
1800
1200
1300
Response
Response
1000
800
600
400
800
300
200
0
-200
-100
-50
0
50
Time
100
150
200
250
s
-200
-100
-50
0
50
Time
100
150
200
250
s
Figure S1: Kinetic analysis of in vitro binding of antibodies to CD16A(158V)
by SPR. The binding of the antibodies (A: hu3F8-IgG1; B: hu3F8-IgG1n; C: hu3F8IgG1-DEL; D: hu3F8-IgG1n-DEL) at various concentrations (from top to bottom: 667,
333, 167, 83, and 42 nM) were measured by Biacore T100. The data were fitted with
Two-State Reaction: A+B=AB=AB*, KD = (kd1/ka1)/(1+ka2/kd2). The colored curves
are experimental data, and the black curves are fitted data. At least three independent
experiments were performed, with one representative set of data was presented.
3
(u g /m l)
c o n c e n tr a tio n
h u Ig G 1
1 0 0 0
h u 3 F 8 -Ig G 1
h u 3 F 8 -Ig G 1 n
1 0 0
h u 3 F 8 -Ig G 1 -D E L
h u 3 F 8 -Ig G 1 n -D E L
1 0
1
S e ru m
0 .1
0 .0 1
0
5 0
1 0 0
T im e
1 5 0
2 0 0
(h )
Figure S2: Blood clearance of the antibodies in nude mice. Quantitation of
serum hu3F8 was carried out by ELISA, and the data were depicted using GraphPad
Prism 6 software. Mean + SEM (n=5). Pharmacokinetic analysis was carried out by noncompartmental analysis of the serum concentration-time data using WinNonlin
software program, and presented in Table 3.
4