1
Supporting text
2
The binding affinity of NtTNF-VHHELP to hTNF in comparison to
3
by surface plasmon resonance (SPR). A detailed analysis of the interaction between TNF and
4
Ec
5
experiments showed that
6
oligomeric TNF, thus recognizing a metatope (epitopes present in both dissociated and
7
polymerized forms). To determine the equilibrium dissociation constant for binding of
8
monomeric TNF to TNF-VHH a Biacore competition experiment, where the binding of
9
soluble TNF to immobilized
Ec
TNF-VHH was measured
TNF-VHH/NtTNF-VHHELP is impaired due to the oligomerization of TNF. Initial binding
Ec
TNF-VHH/NtTNF-VHHELP can bind to both monomeric and
Ec
TNF-VHH was inhibited by soluble TNF-VHH was performed
10
at the lowest TNF concentration possible that still yield reliable binding. A TNF protomer
11
concentration of 386pM resulted in competition curves, which were well described by a
12
monovalent model (Supporting Figure 2A). The equilibrium dissociation constants were
13
derived by non-linear least square fit (KD = 1.30 ±0.098nM for
14
±0.032nM for
15
KD’s were all greater than the used protomer concentration of TNF (c = 386pM) and therefore
16
reliable. Differences observed between the EcTNF-VHH and NtTNF-VHHELP do not necessarily
17
reflect real differences in the equilibrium dissociation constants but may also reflect
18
differences in the concentrations of the protein preparations. Competition assays performed at
19
c(TNF) » KD revealed such concentration differences.
20
Direct binding experiments showed that the antigen binding kinetics of soluble TNF to
21
immobilized
22
with the results from the competition surface plasmon resonance experiments and
23
demonstrated that ELPylation has no negative impact on the VHH paratope. Due to the
24
complexity of the reactions quantitative analysis was not directly possible and would be
25
misleading as the derived values are only valid within the context of an accurate binding
Nt
Ec
Ec
TNF-VHH and KD = 0.590
TNF-VHHELP; for comparison: Certolizumab pegol has a KD 0.110nM). The
TNF-VHH and
Nt
TNF-VHHELP were virtually identical. This is in agreement
1
26
model. The association and dissociation were both biphasic and were not well described by
27
the standard binding models. Therefore, the binding kinetics of
28
VHHELP were compared graphically (Supporting Figure 2B and 2C). The very minor
29
differences are likely artefacts caused by differences in the protein preparations or by partial
30
inactivation as a result of the random covalent coupling.
31
2
Ec
TNF-VHH and
Nt
TNF-
32
Supporting experimental procedures
33
Surface plasmon resonance assays
34
SPR assays were performed at 25°C in HBS-EP buffer (10mM HEPES, 150mM NaCl, 3mM
35
EDTA, 0.05% Tween-20).
36
sensorchips in 10mM sodium acetate pH 4.75 using the EDC/NHS coupling kit (GE
37
Healthcare, Freiburg, Germany). The competition experiments were performed on a
38
BIACore2000 instrument with immobilized
39
capacity (Rmax > 800RU) for
40
thus exhibited an excellent linear dose-response. Samples for competition experiments were
41
incubated for at least 36 h at room temperature prior to the analysis. Dilute TNF and TNF-
42
VHH solutions were stable over these extended incubation times. At low TNF concentrations
43
the interaction can be described by a simple model):
44
monomer
45
Under these conditions binding of soluble monomeric TNF to soluble TNF-VHH directly
46
inhibits binding to immobilized TNF-VHH and the measured binding rates are directly
47
proportional to the concentration of free monomeric TNF. Thus, the equilibrium dissociation
48
constant KD of the interaction can be derived by fitting the binding responses to the equation:
49
TNF  VH H
Rc 0VH H  
Ec
TNF-VHH and
trimeric
monomer
Ec
Nt
TNF-VHHELP were immobilized to CM5
TNF-VHH. The surface had a high binding
TNF and binding was highly mass-transport limited and
KA 
TNF  VH H
Rc 0VH H  0  0
  c VH H  c 0TNF  K D  
0
2  c TNF

c
[ monomer TNF  VH H]
1

monomer
[
TNF]  [VH H] K D
0
VH H
2
 c 0TNF  K D   4  K D  c 0TNF 


50
where KD is the equilibrium dissociation constant, R( c 0VH H =0) the binding response in the
51
absence of the inhibitor (i.e. the TNF-VHH), c 0VH H the concentration of the inhibitor TNF-
52
VHH and c0TNF is the concentration of monomeric TNF.
3
53
The kinetic binding assays were performed on a BIACoreT100 instrument with immobilized
54
Ec
55
The complete absence of mass-transport limitation was verified by injecting TNF at different
56
flow rates. Bound TNF was removed by a 60s injection of 30mM HCl. The TNF-VHH
57
surfaces were stable and less than 10% of activity was lost over 100 regeneration cycles. The
58
double-referenced binding data was evaluated using BIACore Evaluation 4.01, BIACore
59
T100 Evaluation 2.0.1 and MicroCal Origin 5.0.
TNF-VHH and
Nt
TNF-VHHELP with low binding capacity (Rmax < 20 RU) for
60
61
4
trimeric
TNF
62
Supporting Figures
63
Supporting Figure 1. (A) Schematic representation of the plant expression cassettes for
64
Nt
65
peptide; red, TNF-VHH; green, linker sequences; blue, c-myc tag; pink, ER retention signal.
66
(C) Protein sequence of
67
linker sequences; blue, c-myc tag; orange, ELP; pink, ER retention signal. (D) Western blot
68
analysis of transgenic
69
Offspring of high expressors were selected and analyzed by western blot. Different amounts
70
of TSP were loaded on the gel. Recombinant proteins were visualized using anti-c-myc
71
antibodies. Lanes from L1 to L23 - L1, 30ng TSP
72
Nt
73
17/26; L5, 75ng TSP NtTNF-VHHELP 17/26; L6, 150ng TSP NtTNF-VHHELP 17/26; L7, 30ng
74
TSP
75
VHHELP 28/32; L10, 3µg TSP
76
15µg TSP NtTNF-VHH 3/19; L13, 3µg TSP NtTNF-VHH 12/17; L14, 7,5 µg TSP NtTNF-VHH
77
12/17; L15: 15 µg TSP
78
TSP
79
1.5ng NtTNF-VHHELP; L21, 3ng NtTNF-VHHELP; L22, 6ng NtTNF-VHHELP; L23, 15ng NtTNF-
80
VHHELP. ITC purified NtTNF-VHHELP was used as control.
TNF-VHH and
Nt
TNF-VHHELP. (B) Protein-sequence of
Nt
Nt
Nt
TNF-VHH. Black, LeB4 signal
TNF-VHHELP. Black, LeB4 signal peptide; red, TNF-VHH; green,
TNF-VHH plant lines and transgenic
TNF-VHHELP 10/25; L3, 150ng TSP
Nt
Nt
Nt
Nt
TNF-VHHELP plant lines.
TNF-VHHELP 10/25; L2, 75ng TSP
TNF-VHHELP 10/25; L4, 30ng TSP
TNF-VHHELP 28/32; L8, 75ng TSP
Nt
Nt
Nt
Nt
TNF-VHH 12/17; L16: 3 µg TSP
Nt
TNF-VHHELP
TNF-VHHELP 28/32; L9, 150ng TSP
TNF-VHHELP 3/19; L11, 7.5µg TSP
TNF-VHH 13/11; L18, 15µg TSP
Nt
Nt
Nt
Nt
TNF-
TNF-VHH 3/19; L12,
TNF-VHH 13/11; L17, 7.5µg
TNF-VHH 3/19; L19, 0.5ng NtTNF-VHHELP; L20,
81
82
Supporting Figure 2
83
Surface plasmon resonance analysis of the antigen binding properties of the EcTNF-VHH
84
and
85
logarithm of the total TNF-VHH concentration and the equilibrium dissociation constants for
86
Nt
NtTNF-V H
H ELP.
(A) The binding rates of free soluble hTNF were plotted against the
TNF-VHHELP (black squares),
Ec
TNF-VHH (grey circles) were determined by non-linear
5
Nt
87
least square fit (solid lines). The binding kinetics of soluble hTNF to immobilized
88
VHHELP (B) and EcTNF-VHH (C). Shown are the double referenced binding curves for a 2-fold
89
serial dilution series starting from a protomer hTNF concentration of 237nM.
90
6
TNF-