Supplemental Information
Supplemental Materials and Methods
Expression and purification of PF-05082566
PF-05082566 was expressed in human 293F cells using the Freestyle 293 expression system
(Invitrogen, Carlsbad, CA). Briefly, 1 mg of purified DNA is used to transfect 1 L of 293F cells
that are at a concentration of 1 x 106 cells/ml. Six days after transfection, the supernatants are
harvested and the antibody is purified using a HiTrap protein A column (GE Healthcare,
Piscataway, New Jersey) with elution with 20 mM Sodium Acetate pH 3.5. PF-05082566 was
then dialyzed into 20 mM Sodium Acetate, 120 mM NaCl, pH 5.5 and stored at 4°C. The
concentration of each mAb was determined using absorbance at 280. To assess purity, both
non-reducing and reducing SDS-PAGE were performed using 4-12% NuPAGE gels (Invitrogen,
Carlsbad, CA) in MES buffer. Analytical size exclusion chromatography (SEC) analyses were
performed on a Waters 2695 HPLC (Milford, MA) employing a TSK gel Super SW3000 (7.8
mm x 300 mm, Tosoh Bioscience, Montgomeryville, PA) column. The column was operated at
a flow-rate of 0.5 ml/min with a mobile phase of 0.2 M Di-Sodium Phosphate pH 7.2 with 25 g
antibody injected for each sample.
Kinetic studies using SPR Biosensor technology
Kinetic rate constants for binding of PF-05082566 to 4-1BB were measured by surface plasmon
resonance (SPR) using a Biacore 3000 instrument (GE Healthcare, Piscataway, NJ). Binding
experiments were carried out at 25 oC in a running buffer comprising 150 mM NaCl, 25mM
HEPES pH 8.0, 6 mM MgCl2, 0.005% polysorbate 20, 0.5mM sodium azide. Standard amine
coupling chemistry was used to immobilize PF-05082566, recombinant human and cyno 4-1BB
Fc-fusion proteins on a CM5 sensorchip (GE Healthcare, Piscataway, NJ) using a 0.1 mg/mL
solution in 10 mM sodium acetate pH 5.0. The human and cyno 4-1BB was injected over
immobilized PF-05082566 at a range of concentrations (80 nM to 0.16 nM,) at a 50 mL/minute
flow rate, for 3.6 minutes followed by a 26 minute dissociation period using the Kinject feature
of the Biacore 3000 instrument. PF-05082566 Fab was injected over immobilized human and
cyno Fc-fusion proteins at 500nM to 1 nM, at a flow rate of 50 mL/minute flow rate, for 1
minute followed by a 26 minute dissociation period using the Kinject feature of the Biacore 3000
instrument. The bound complex was regenerated by a 1 minute pulse of 10 mM phosphoric acid
in water. Data analysis was performed using the Scrubber2 software (BioLogic Software,
Campbell, Australia). Sensograms were fit to a simple 1:1 Langmuir binding model.
Isothermal tritration calorimetry
ITC experiments were carried out on a VP ITC instrument (GE Healthcare) at 25 oC. Protein
samples were exchanged into a buffer containing 150 mM NaCl, 25 mM HEPES, pH 7.5, by size
exclusion chromatography (superdex 200) and the concentration determined
spectrophotometrically using a predicted E280 of 8480 M-1 cm-1 for the recombinant cyno 4-1BB
and 204000 M-1 cm-1 for the PF-05082566 IgG. In a typical experiment, 19 x 15 µL injections of
100 µM cyno 4-1BB were made in to a 10 µM binding sites of IgG. Heats of dilution were
determined from the saturation end point. Data from the direct binding experiments were
analyzed using the ORIGIN software (OriginLab) and fit to a simple 1:1 equilibrium binding
model.
4-1BB Binding ELISA
Human 4-1BB IgG1Fc chimera (R&D Systems, Minneapolis, MN) was resuspended with
Dulbecco’s Phosphate Buffered Saline (DPBS) containing 0.1% bovine serum albumin (BSA) to
0.2mg/ml and diluted with DPBS to a final concentration of 0.03 ug/ml. Nunc-Immuno Maxisorp
96 well plates were coated with 0.1ml per well of the recombinant 4-1BB chimera leaving empty
wells for nonspecific binding controls and incubated at 4oC overnight. The 4-1BB solution was
removed and plates washed with wash buffer (0.05% Tween-20 in DPBS). Blocking buffer (5%
non-fat dry milk, 0.05% Tween-20 in DPBS) was added to all wells and incubated at 4oC for 1h
with mixing. The blocking buffer was removed and plates washed with wash buffer. Serial
dilutions of the 4-1BB test antibodies were prepared in DPBS and diluted Ab was added to the
plates. Plates were inucbated 1.5h at room temperature. Antibody solution was removed and the
plates washed with wash buffer. Horseradish peroxidase labeled goat anti-human IgG, F(ab’)2
specific F(ab’)2 antibody (Jackson Immunoresearch, West Grove, PA) was diluted with DPBS
and added to the plates. The plates were incubated 1h at room temperature and washed with
wash buffer. SureBlue TMB microwell peroxidase substrate (Kirkegaard & Perry Labs
Gaithersburg, MD) was added and incubated for 20 minutes at room temperature. The reaction
was stopped by the addition of an equal volume of 2M H2 SO4 and absorbance was read at 450
nm on a Molecular Devices Spectra Max 340 (Molecular Devices, Sunnyvale, CA).
Flow cytometry assessment of antibody binding to cell surface 4-1BB
4-1BB expressing cell lines or activated primary peripheral blood mononuclear cells (PBMC)
were used to assess antibody binding on both the human and cynomolgus 4-1BB receptors. Cells
were harvested and washed (~5 x 105 cells/tube) using cold Fluorescence Activated Cell Sorting
(FACS) wash buffer (BD Biosciences, San Jose, CA). Next, 0.1 ml of various concentrations of
Alexa Fluor-647 labeled antibody was added to the cells (starting at 155 g/ml and using a 10-
fold titration) diluted in wash buffer containing 0.005 mg/ml of cytocholasin B (Sigma, St.
Louis, MO). The cells were gently mixed at room temperature for 3 hours. Next, the cells were
washed twice and resuspended in 0.3 ml/tube with cold phosphate buffered saline (PBS)
containing 2% paraformaldehyde. 10,000 events per sample were collected and analyzed using a
Becton Dickinson FACSCalibur and FlowJo 8.8.2 software (TreeStar Inc. Ashland, OR).
Antibody 4-1BB ligand competition
Antibodies were tested for their ability to block the binding of the human 4-1BB_IgG1Fc
chimera to plate bound recombinant 4-1BB ligand (Invitrogen, Carlsbad CA). Recombinant
human 4-1BB ligand (4-1BBL) was resuspended to 0.2 mg/ml in DPBS + 0.1% BSA and then
diluted to 1 g/ml in DPBS. Nunc-Immuno MaxiSorp surface 96 well plates were coated with
0.1 ml/well of the 4-1BBL solution overnight at 4oC. The following day the 4-1BBL solution
was removed and 0.2 ml blocking buffer (1% BSA, 0.05% Tween-20 in DPBS) added and
incubated at room temperature for 2 hours. During the blocking step the antibody stocks were
diluted in a range from 8 ng/ml to 6 g/ml in DPBS. Recombinant human 4-1BB_IgG1Fc was
resuspended to 0.2 mg/ml in DPBS + 0.1% BSA and then diluted to 0.02 g/ml in DPBS. The
blocked 4-1BBL coated plates were washed three times with 0.2 ml wash buffer (0.05% Tween
20 in DPBS). 60 l antibody dilutions were added along with 60 l 4-1BB_IgG1Fc chimera and
incubated at room temperature 1.5 hours. Plates were washed as previously described.
Horseradish peroxidase anti-6xHistidine tag antibody (R&D Systems, Minneapolis, MN) was
diluted 1:1000 in DPBS, 50 l of the resulting solution added to the wells of the washed plates,
and incubated at room temperature 1 hour. Plates were washed as previously described. 50 l
TMB substrate solution was added to each well and incubated at room temperature for 20
minutes. The reaction was stopped with 50 l 0.2N H2SO4 and absorbance at 450 nm read using
a Molecular Devices plate reader.
Supplemental Figure Legends
Supplemental Figure 1: FACS data corresponding to Figure 1A and Figure 1B, Histogram plots
demonstrating staining of human 4-1BB expressing 300-19 at 100nM and 10nM Alexa-fluor 647
conjugated antibody. Staining of cells by PF-05082566 (open histogram) is shown with staining
of cells by an Alexa-fluor 647 labeled isotype control antibody at the corresponding
concentration (black histogram). FL-4 Geometric Mean Fluorescence is represented in the
histogram legend.
Supplemental Figure 2: FACS data corresponding to Figure 1C and Figure 1D, Forward
Scatter/Side Scatter gating was used to identify live PBMC from 72h PHA stimulated human and
cynomolgus monkey PBMC and subsequently gated on CD3+ cells. The Geometric mean
fluorescence of the CD3+ population was used to demonstrate specific staining of cells with
Alexa-fluor 647 labeled PF-05082566 versus staining with an Alexa-fluor 647 labeled human
IgG2 isotype control at various concentrations. Representative data for both human and
cynomolgus monkey PBMC staining at 100uM labeled antibody is shown. Histogram overlays
compare staining with PF-05082566 (open histogram) versus staining with an isotype control
(shaded histogram).
Supplemental Figure 3: PF-05082566 parental mAbs inhibit the growth of colorectal carcinoma
and melanoma in vivo. (A) Mean tumor volume at time points following subcutaneous
administration of three million LoVo carcinoma cells plus 1.5 million human PBMC in the right
flank of SCID Bg mice (n = 8 animals/group). (B) The volume of each individual tumor on the
final study day (day 21) following subcutaneous administration of three million WM-266
melanoma cells plus 1.5 million human PBMC in the right flank of SCID Bg mice (n = 6
animals/group). Animals were randomized based on tumor size on Day 7 post implantation and
given a single dose of the indicated concentrations of PF-05082566 non germlined parental mAb
of the indicated isotype or control. For panel B, all animals were dosed with 1 mg/kg of the
indicated mAb. Tumors were measured two to three times per week until study termination.
Statistical significance was determined using Student’s t-test (*p<0.05, **p<0.005).