Supplementary Information
Development of a High Affinity, Non-covalent
Biologic to Add Functionality to Fabs
Kendra N. Avery, Cindy Zer, Krzysztof P. Bzymek
and John C. Williams*
Department of Molecular Medicine,
Beckman Research Institute at City of Hope
Materials and Methods:
Protein Expression and Purification
Protein L and meditope-protein L fusion protein DNA coding sequences were
inserted between BamH1 and Xho1 sites of a modified pET28b vector encoding
an N-terminal 6-His-tagged SMT3. Protein expression was induced with 200 M
final concentration IPTG in BL21 (DE3) E. coli in LB media under Kanamycin
selection for 4 hrs at 37 C. Cells were lysed by french pressure cell, clarified by
centrifugation, filtered through 0.22 m filter and loaded onto a Ni-NTA column
(Thermo Scientific HisPur). After washing with 10 mM imidazole in PBS, the
protein was eluted with 250-500 mM imidazole. The His6-SMT3 fusion was
cleaved by ULP1-His6 protease with 10-20 mM DTT while being dialyzed into
PBS at 4C. The cleaved protein was further purified from ULP1 and SMT3 by
reverse Ni-NTA and concentrated to 2.5-3 mL. The concentrated product was
applied to a Superdex G75 preparative column (GE Healthcare) for final
purification. The final product was concentrated, aliquot into small volumes and
flash frozen. Protein aliquots were stored at -80 C. Each mutant construct of the
meditope-proL fusion, protein L and MPL6-GFP were purified in a similar
manner. Meditope peptide was synthesized at the City of Hope Synthetic
Biopolymer Chemistry Core by solid phase synthesis on a semi-automated
peptide synthesizer, purified by HPLC and confirmed by mass spectrum analysis.
Prescription grade parental trastuzumab and cetuximab were obtained from the
City of Hope Pharmacy and used without additional purification. The extracellular
portion of sHER2 (sErbB2) was expressed in High 5™ insect cells and purified
by Ni-NTA affinity and size exclusion chromatography as previously described 1.
Meditope-enabled trastuzumab was expressed in NS0 cells and purified by
protein A affinity chromatography and size exclusion gel chromatography as
previously described1.
Sequences
(S) is not counted in the numbering scheme as it is a residual amino acid from
the cleavage of the His6-SMT3 tag by ULP1 protease.
MPL6
(S)CQFDLSTRRLKCGGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYA
ALLAKVNGEYTADLEDGGNHMNIKFAG
meditope
CQFDLSTRRLKC
protein L
(S)EVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGG
NHMNIKFAG
MPL6 variant F3A R8A
(S)CQADLSTARLKCGGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYA
ALLAKVNGEYTADLEDGGNHMNIKFAG
MPL6 variant Y51W L55H
(S)CQFDLSTRRLKCGGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRWA
ALHAKVNGEYTADLEDGGNHMNIKFAG
MPL no linker
(S)CQFDLSTRRLKCVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVN
GEYTADLEDGGNHMNIKFAG
MPL3 (GSE)
(S)CQFDLSTRRLKCGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLA
KVNGEYTADLEDGGNHMNIKFAG
MPL9 (GSGSGGGSE)
(S)CQFDLSTRRLKCGSGSGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAY
RYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL3 (PSE)
(S)CQFDLSTRRLKCPSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLA
KVNGEYTADLEDGGNHMNIKFAG
MPL3 (PPE)
(S)CQFDLSTRRLKCPPEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLA
KVNGEYTADLEDGGNHMNIKFAG
MPL3 (PPP)
(S)CQFDLSTRRLKCPPPVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLA
KVNGEYTADLEDGGNHMNIKFAG
MPL6 Pro (PPPPPP)
(S)CQFDLSTRRLKCPPPPPPVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAA
LLAKVNGEYTADLEDGGNHMNIKFAG
MPL6 (PPPGPP)
(S)CQFDLSTRRLKCPPPGPPVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAA
LLAKVNGEYTADLEDGGNHMNIKFAG
MPL6 (GGPGSE)
(S)CQFDLSTRRLKCGGPGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAA
LLAKVNGEYTADLEDGGNHMNIKFAG
MPL6-GFP
(S)CQFDLSTRRLKCGGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYA
ALLAKVNGEYTADLEDGGNHMNIKFAGGGSGGSMVSKGEELFTGVVPILVELD
GDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFS
RYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
KGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD
HYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMD
ELYK
SPR Measurements
Protein concentrations were calculated by measuring the absorbance at 280 nm
using extinction coefficients estimated by Expasy Protparam tool. All SPR
measurements were performed at 25 °C on the GE Biacore T100 instrument
using Series S CM5 sensor chips. All ligands (trastuzumab, cetuximab, memAb
trastuzumab or sHER2) were amine coupled using EDC/NHS chemistry in GE
acetate buffer pH 5.5 at densities suitable for kinetics experiments using the
equation:
RL=Rmax X (ligand MW/analyte MW) X 1/Sm
(Rmax values between 50-150 RU were used and typically 10% more ligand was
immobilized to account for non-productive coupling orientations). Typical
experiments with MPL variants as analyte had memAb trastuzumab immobilized
at ~1200 RU and meditope as analyte ~5000 RU. Surface densities of 300-700
RU were necessary to achieve Rmax signals from 50-150 for the immobilization
of HER2 binding to MPL(GFP)/Fab experiments. The running buffer for all
experiments was GE buffer HBS-EP+ (10 mM Hepes pH 7.4, 150 mM NaCl, 3
mM EDTA and 0.05% v/v surfactant P20). Kinetics experiments were carried out
at a flow rate of 30 L/min using HBS-EP+ as both running and regeneration
buffer in the case of the meditope binding experiments. For the MPL to memAb
and Fab binding to HER2 experiments, 10 mM glycine pH 2.0, 1 M NaCl was
used as regeneration buffer, followed by a wash with HBS-EP+. Due to the
stringent regeneration condition, experiments could not all be carried out on the
same chip due to ligand damage over time, therefore replicate experiments were
frequently performed on different chips with similar coupling densities. This ligand
damage over time due to the pH 2.0 regeneration is likely the reason for the
relatively high standard deviation values on some of our measurements in
addition to the lack of curvature in the off rate sensograms. In the experiments
with memAb trastuzumab Fab and memAb trastuzumab Fab + 500 nM MPL6,
the same concentrations of Fab were used in each experiment, but 500 nM
MPL6 remained constant in each of the memAb Fab with MPL samples. The
0.625 concentration was removed from the data fit in figure 3B due to an air
bubble. Figure 3 data were performed on the same chip for direct comparison
and figure S4 shows data collected from different chips. For the memAb Fab with
MPL6-GFP experiments, the memAb Fab concentration was varied while MPL6GFP was held constant at 100 nM in each sample. Kinetic parameters were
calculated using Biacore T100 Evaluation software version 2.0.1 using the 1:1
binding model. Kinetic parameters were calculated for each individual experiment
and the averages and standard deviations of replicate measurements are
reported.
Alexa Fluor labeling of MPL6 and mAbs
Parental and memAb trastuzumab were labeled with Alexa Fluor 488 according
to manufacturer's protocol (Invitrogen). MPL6 was labeled at 7 mg/ml with ten
fold more Alexa Fluor 647 than recommended in the protocol and three hours of
labeling reaction at room temperature to account for the inefficiencies in labeling
a small protein.
FACS Analysis
SKBR3 cells were maintained in 10% FBS-supplemented DMEM at 37 C and
5% CO2 humidifed atmosphere. Each sample contained 1 million SKBR3 cells.
Cells were trypsinized and washed once with 1 mL wash buffer (0.3% BSA in
PBS), and then incubated with 100 L of 10 nM either mAb and 50 nM of MPL6
for 30 min on ice. Alternatively, cells were first incubated with 100 L of 10 nM
either mAb for 15 min, washed once with 1 ml wash buffer, then another 15 min
with 100 L of 50 nM MPL6 on ice. At the end of the incubation, the cells were
washed twice with 1 mL wash buffer, and resuspended in 1 mL of wash buffer
supplemented with SYTOX® Blue dead cell stain (Invitrogen). FACS were
performed on a CyAn™ ADP Analyzer (Beckman Coulter), and the data were
analyzed with Flowjo software.
Microscopy
50,000 SKBR3 cells were seeded onto cover slips in 12 well plates over night.
The culture medium was replaced with 250 L of medium containing 50 nM
Alexa Fluor 555 labeled-memAb or parental trastuzumab and 250 nM MPL6-GFP
at 37°C for 30 min. The cells were washed twice with pre-warmed PBS, fixed
with room temperature 3.7% formaldehyde diluted in PBS, washed twice again
with room temperature PBS, and then mounted with ProLong® Gold Antifade
Reagent with DAPI (Invitrogen) onto glass slides. Images were taken with
Olympus IX81 automated inverted microscope at 64 x magnification. Images
were analyzed with Photoshop.
Supplemental Figures and Tables:
Supplemental Figure 1 Representative SPR sensograms with residuals showing
binding avidity of A) MPL6 and the loss of avidity for B) monomeric meditope, C)
protein L, D) MPL F3A R8A and E) MPL Y51W L55H with immobilized memAb
trasstuzumab IgG. Analyte concentrations include A) 10 nM - 78 pM, B) 16 µM 62.5 nM, C) 1 µM - 15.6 nM, and D-E) 2 µM -20 nM.
Supplemental Figure 2 Representative SPR sensograms with residuals showing
the binding of A) MPL no linker, B) MPL3 (GSE), and C) MPL9 (GSGSGGGSE)
to immobilized memAb trastuzumab. Analyte concentrations include 78 pM - 10
nM.
F.
10
Response Units
Response Units
E.
5
0
0
400
600
10
0
0
800
200
10
0
-10
-20
400
600
800
Time (s)
20
Residuals
Residuals
200
Time (s)
20
20
10
0
-10
-20
0
200
400
600
800
0
200
400
600
800
Supplemental Figure 1 Representative SPR sensograms with residuals showing
the binding interaction of A) MPL3 (PSE), B) MPL3 (PPE), C) MPL3 (PPP), D)
MPL6 (PPPPPP) E) MPL6 (PPPGPP) and F) MPL6 (GGPGSE) with immobilized
meditope-enabled trastuzumab. Analyte concentrations include 78 pM to 10 nM
for A-C, E-F and 2.5 nM - 320 nM for D).
A.
B.
Response Units
80
0
0
200
400
Time (s)
400
600
Residuals
Residuals
200
15
-15
C.
600
20
10
100
200
Time (s)
300
400
0
100
200
300
400
memAb Fab + MPL6-GFP to HER2
Response Units
20
0
2
-2
D.
MPL-GFP to memAb
Response Units
40
0
0
Residuals
memAb Fab + MPL6 to HER2
80
40
0
0
0
400
4
-4
0
400
800
Time (s)
1200
800
1200
Residuals
Response Units
memAb Fab to HER2
160
0
200
0
200
20
-20
400
Time (s)
400
600
600
Supplemental Figure 2 Representative SPR sensograms with residuals showing
the binding of A) memAb Fab with immobilized HER2, B) memAb Fab preincubated with excess MPL6 to immobilized HER2, C) MPL6-GFP to immobilized
memAb and D) memAb Fab pre-incubated with excess MPL6-GFP with
immobilized HER2. Analyte concentrations of memAb Fab in A-B and D include
78 pM - 10 nM, MPL6 remained constant at 500 nM in all dilutions for B) and
MPL6-GFP remained constant at 100 nM in all dilutions for D). MPL6-GFP
concentrations in C) were 10 nM - 78 pM.
Supplemental Figure 3 Representative SPR sensogram with residuals showing
the binding interaction of MPL6 with immobilized parental trastuzumab. Analyte
concentrations include 7.8 nM - 2 µM.
Supplemental Table 1. Summary of dissociation and kinetic constants calculated
from SPR analysis for engineered IgG binding proteins with memAb and memAb
Fab complexed to IgG binding proteins with antigen.
Analyte
Immobilized
Ligand
kon
(X 105)
koff
(X 10-4)
KD
(X 10-10)
MPL6
memAb
trastuzumab
7.9 +/- 3.2
1.8 +/-1.6
1.8 +/- 0.4
MPL6-GFP
memAb
trastuzumab
3.1 +/- 3.7
1.9 +/- 1.1
13 +/- 8
memAb Fab
HER2
14 +/- 6
2.4 +/- 1.1
1.9 +/- 1.0
memAb Fab +
MPL6
HER2
14 +/- 1
1.9 +/- 0.5
1.3 +/- 0.4
memAb Fab +
MPL6-GFP
HER2
6.5 +/- 2.7
3.1 +/- 1.8
4.5 +/- 0.7
References
1) J. M. Donaldson, C. Zer, K. N. Avery, K. P. Bzymek, D. A. Horne, J. C.
Williams, Proc Natl Acad Sci U S A 2013, 110, 17456-17461.