1

Supplementary Information

A heterodimeric Fc-based bispecific antibody simultaneously targeting VEGFR-2 and Met

exhibits potent anti-tumor activity

Hye-Ji Choi, Ye-Jin Kim, Sangho Lee, and Yong-Sung Kim

Inventory of Supplementary Information

Supplementary Data :

5 supplementary figures

4 supplementary tables

Supplementary Materials and Methods

Supplementary References

2

Supplementary Figure S1. Sequence alignment of CH3 domain of human IgG isotype antibodies

(hIgG1, hIgG2, hIgG3, hIgG4) with highlights of the mutated residues in EW–RVT heterodimeric Fc

variant (K360E/K409WCH3A–Q347R/D399V/F405TCH3B) in comparison with the reported KiH

(T366S/L368A/Y407VCH3A–T366WCH3B) (1, 2), DD–KK (K409D/K392DCH3A–D399K/E356KCH3B)

(3), and HA–TF (S364H/F405ACH3A–Y349T/T394FCH3B) heterodimeric Fc variants, as indicated with

each color code. The inverted arrows on the top indicate the 23 residues located in the homodimeric

CH3-CH3 interface within the distance limit of 4.6 Å, analyzed using the human IgG1 Fc crystal

structure (PDB code 3AVE) (4). The amino acids are numbered by the EU nomenclature (5).

3

Supplementary Figure S2. A, schematic presentation of mammalian expression plasmids encoding

a scFv-FcCH3A carrying one CH3 variant (indicated as CH3A) and a FcCH3B carrying the other CH3

variant (indicated as CH3B) used in this study. The Fc region of human IgG1 includes the hinge-

CH2-CH3 regions (residues 225 – 447 in EU number). PCMV, CMV promoter; H, human IgG1 hinge

sequence; CH2, human IgG1 CH2 sequence; CH3A/B, human IgG1 CH3 variant sequence; stop,

translational stop sequence. The positions of enzymes sites used for the subcloning are indicated.

B, western blotting analysis under reducing conditions of the purified scFv-FcCH3A/FcCH3B proteins

carrying the indicated CH3 variant pair. The proteins were prepared and detected as described in Fig.

2C. The arrows indicate the position of scFv-FcCH3A (~53.5 kDa) and FcCH3B (~26.3 kDa). C,

western blotting analysis under non-reducing conditions of the purified scFv-FcCH3A and FcCH3B

proteins carrying the indicated CH3 variant pair, independently expressed in HEK293F cell cultures.

The respective plasmid encoding the scFv-FcCH3A or FcCH3B was singly transfected into HEK293F

cells and the cells were cultured for 6 days before protein purification from the culture supernatant

using protein A resin. The arrows indicate the assembled scFv-FcCH3A homodimer (~103.2 kDa) and

FcCH3B homodimer (~52.3 kDa).

4

Supplementary Figure S3. Representative SPR sensograms show the kinetic interactions of msMet,

tanibirumab, and bsVeMet antibodies with the antigens, Met and VEGFR-2. Serially 2-fold diluted

concentrations (from 400 to 3.13 nM as color-code indicated) of msMet, bsVeMet, and tanibirumab

antibodies were flowed over Met-Fc- or VEGFR-2-Fc-immobilized surface at ~1,000 RUs.

Quantitative kinetic interaction parameters are shown in Supplementary Table S4.

5

Supplementary Figure S4. A, binding profiles of the msMet, tanibirumab and bsVeMet antibodies

(each 100 nM) to cell surface-expressed Met or VEGFR-2 in HUVEC and MKN45 cells, monitored

by flow cytometry. The analysis with commercial anti-Met and anti-VEGFR-2 antibodies (positive

controls) confirmed the significant Met and VEGFR-2 expression in HUVECs and only Met

expression with negligible VEGFR-2 expression in MKN45 cells. B, effects of the antibodies on the

proliferation of the endothelial HUVECs stimulated by single ligand HGF or VEGF alone, monitored

by MTT assay. HUVECs were treated with medium (control) or the indicated antibodies (0.1 and

0.5 μM) for 42 h in the presence of 50 ng/mL HGF or 20 ng/mL VEGF prior to the MTT assay.

Percentage of cell viability was evaluated versus the respective control. Data shown represent the

mean ± SD (error bars) of 4 independent experiments performed in duplicate. *P < 0.05 vs. the

monospecific antibodies group. C, western blotting analysis of the downstream signaling of

VEGFR-2 and/or Met in HUVECs, serum-starved for 6 h and then stimulated for 10 min with HGF

(10 ng/mL) or VEGF (10 ng/mL) prior to treatments for 1 h with medium (control) or with the

indicated antibodies (each 1 μM). In (B and C), the combined treatments of msMet and tanibirumab

at the equivalent molar ratio are indicated as M+T.

6

Supplementary Figure S5. A, representative photomicrographs showing the effects of antibodies

on the tube formation of HUVECs stimulated by treatments of HGF and/or VEGF, as described in Fig.

5C. HUVECs were plated onto matrigel-coated plates and incubated with PBS (control), HGF (200

ng/mL), and/or VEGF (200 ng/mL) in the absence (Upper panels) and presence (Lower panels) of the

antibodies (each 1 μM) for 4 h, prior to the picture taken. Images are representative of two

independent experiments. Image magnification, ×100; scale bar, 50 μm. B, quantification of

HUVEC tube formation, the images of which are shown in Fig. 5C. The tube formation was

quantified by measuring tubule extensions from endothelial cell bodies, to determine tubule length (6),

using Image pro plus (Media Cybermetics, USA). Tubule length was measured as an average of

three fields of view in each well for the two independent experiments. Error bars, ± SD. **P <

0.005. In (A and B), the combined treatment with msMet and tanibirumab at the equivalent molar

ratio are indicated as M+T.

7

Supplementary Table S1. CH3 variant pairs tested in the scFv-FcCH3A/FcCH3B system for the

heterodimeric Fc formation.a

Variants CH3A (scFv-Fc) CH3B (Fc)

E-R K360E Q347R

W-VT K409W D399V/F405T

EW-RVT K360E/K409W Q347R/ D399V/F405T

KiH (Genentech) T366S/L368A/Y407V (Hole) T366W (Knob) a The residue numbering is according to the EU index as in Kabat (5).

8

Supplementary Table S2. Purification yields of Fc proteins obtained from transient expression in

HEK293F cellsa.

Fc proteins Purification yield b

( mg/100-mL of transfected cells)

Fc-WT 2.9 ± 0.6

Fc-EW–RVT 2.9 ± 0.3

Fc-KiH 2.6 ± 0.5 a The two Fc plasmids carrying the CH3 wild type (WT) or variant pairs were co-transfected at the

equivalent molar ratio into HEK293F cells in 100-mL culture volume following the standard protocol

(7). After 6 day culture, Fc proteins were purified from the cell culture supernatant using protein A

affinity column.

bThe values represent mean ± SD of four independent experiments.

9

Supplementary Table S3. The kinetic parameters for the interactions of Fc proteins with FcRn and

FcγR proteins, determined by SPRa.

Proteins Fc proteins kon (M-1s-1) koff (s

-1) KD (M)

FcRn (pH6.0)

Fc-WT 6.69±0.38×104 1.75±0.09×10

-3 2.61±0.46×10

-8

Fc-EW–RVT 2.10±0.28×104 9.52±0.46×10

-4 4.54±0.16×10

-8

Fc-KiH 3.40±0.67×104 7.50±0.85×10

-4 2.20±0.13×10

-8

FcγRIIa

Fc-WT 9.26±1.73×104 2.01±0.11×10

-1 2.17±0.65×10

-6

Fc-EW–RVT 1.53±0.12×105 2.12±0.06×10

-1 1.38±0.51×10

-6

Fc-KiH 4.57±0.39×105 7.32±0.28×10

-1 1.60±0.71×10

-6

FcγRIIIa

Fc-WT 6.31±0.54×104 1.18±0.05×10

-2 1.87±0.09×10

-7

Fc-EW–RVT 2.93±0.07×104 6.68±0.11×10

-3 2.28±1.54×10

-7

Fc-KiH 2.55±0.29×105 4.88±0.31×10

-2 2.17±0.14×10

-7

FcγRIIIb

Fc-WT 6.77±0.56×104 9.50±0.41×10

-2 1.40±0.73×10

-6

Fc-EW–RVT 5.06±0.41×104 1.58±0.04×10

-1 3.13±1.17×10

-6

Fc-KiH 3.65±0.53×104 1.19±0.06×10

-1 3.26±1.13×10

-6

a Each value represents the mean ± SD of two independent experiments. In each experiment, at least

5 data sets were used in the determination of the kinetic constants. The representative sensograms

are shown in Fig. 3F for FcRn and Fig. 3G for FcγR proteins.

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Supplementary Table S4. Kinetic binding parameters for the interactions of the antibodies with the

antigens, Met or VEGFR-2, monitored by SPRa.

Antibodies antigens kon (M-1s-1) koff (s

-1) KD (M)

msMet Met 2.15±0.09×104 1.95±0.14×10

-4 9.09±0.15×10

-9

bsVeMet Met 4.17±0.02×10

4 1.50±0.26×10

-4 3.59±0.13×10

-9

VEGFR-2 6.53±0.40×104 1.16±0.23×10

-3 1.78±0.06×10

-8

Tanibirumab VEGFR-2 4.34±0.22×105 2.79±0.19×10

-4 6.42±0.86×10

-10

a Each value represents the mean ± SD of two independent experiments. In each experiment, at least

5 data sets were used in the determination of the kinetic constants. The representative sensograms

are shown in Supplementary Fig. S3.

11

Supplementary Materials and Methods

Reagents

Recombinant human proteins of FcγRIIa, FcγRIIIa, FcγRIIIb, and human IgG1 Fc-fused extracellular

domain of Met (Met-Fc) were purchased from R&D Systems. Human FcRn protein was purchased

from Sino Biological Inc. Recombinant VEGF165 and HGF proteins were from Promokine

(Heidelberg, Germany). Anti-VEGFR-2 mAb tanibirumab (8) and human IgG1 Fc-fused

extracellular domain of VEGFR-2 (VEGFR-2-Fc) were provided from PharmAbcine (Daejoen,

Korea). Mouse anti-Met, rabbit anti-phospho Met (Tyr1234/1235), rabbit VEGFR-2, rabbit anti-

phospho VEGFR-2 (Tyr1059), rabbit anti-MAPK, rabbit anti-phospho-p44/42 MAPK

(Thr202/Tyr204), rabbit anti-Akt and rabbit anti-phospho-Akt (Ser473) antibodies were from Cell

signaling Technology (Danvers, MA). Rabbit anti-β actin antibody was from Santa Cruz

Biotechnology (Santa Cruz, CA). Other reagents were analytical grade.

Design and evaluation of heterodimeric Fc

We identified candidate mutation pairs at the CH3-CH3 interface (3) and evaluated for the feasibility

to stabilize Fc heterodimerization and destabilize Fc homodimerization based on the structural

analysis of the 2 Å human IgG1 Fc crystal structure (PDB code 3AVE) (4). Each substitution was

modeled in the 3AVE structure by optimizing side-chain conformations of the mutated residue and its

surrounding residues in PyMol software (DeLano Scientific LLC).

Fc heterodimerization was screened using the scFv-FcCH3A/FcCH3B expression system (Fig.

2A) (3). The human IgG1 Fc containing the hinge-CH2-CH3 (residues 225 - 447 in EU numbering

(5)) was subcloned into the mammalian expression vector pcDNA 3.1 (Invitrogen), generating a

FcCH3B expression plasmid (Supplementary Fig. S2A). The human AY4 scFv specifically

recognizing human death receptor 4 (9) was in frame subcloned into the N-terminus of human IgG1

Fc, generating scFv-FcCH3A expression plasmid (Supplementary Fig. S2A). DNAs encoding the

designed CH3 variant pair of CH3A and CH3B were synthesized (Bioneer, Korea) and subcloned into

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XhoI/BamHI sites of the scFv-FcCH3A (hAY4 scFv-hinge-CH2-CH3A) and FcCH3B (hinge-CH2-CH3B)

plasmids (Fig. 2B and Supplementary S2A).

Construction, expression, and purification of antibodies

For the expression in FcCH3A/FcCH3B heterodimer, EW–RVT and KiH CH3 variant pair was subcloned

using XhoI/BamHI into the corresponding CH3 region of Fc in pcDNA3.1 plasmid (Supplementary

Fig. S2A), generating two plasmids encoding Fc-EW–RVT proteins (hinge-CH2-CH3A (EW) ×

hinge-CH2-CH3B (RVT)) (Fig. 3A).

For the construction of the msMet antibody in the format of Fv-Fc (VH-hinge-CH2-CH3A

(EW) × VL-hinge-CH2-CH3B (RVT)), the variable heavy (VH) and light (VL) sequence from

MetMab (OA5D5, Genentech) (10, 11) was synthesized and subcloned into the N-terminus of Fc

heterodimer (Fig. 4A). For the generation of anti-Met × VEGFR-2 bsVeMet antibody in the format

of scFv-Fc (anti-Met scFv-hinge-CH2-CH3A (EW) × anti-VEGFR-2 scFv-hinge-CH2-CH3B (RVT)),

the anti-Met and VEGFR-2 scFv antibodies, generated by linking VH and VL sequences derived from

the anti-Met OA-5D5 (10, 11) and anti-VEGFR-2 tanibirumab (8), respectively, with a (G4S)3 peptide

linker, were subcloned into the N-terminus of Fc heterodimer (Fig. 4A).

Each pair of the two plasmids encoding Fc heterodimer, msMet and bsVeMet antibodies was

transiently co-transfected at the equivalent molar ratio into HEK293F cells in FreeStyle 293 media,

ranged from 100-ml to 400-ml liter culture volume, following the standard protocol (7). After 6

days of cultures, the antibodies were purified from the culture supernatants as described above and

extensively dialyzed to resuspend in PBSG buffer (PBS plus 10% glycerol, pH 7.4). The antibodies

concentrations were determined using a BCA protein assay (Pierce).

SEC analysis

SEC analysis was performed on the Agilent 1100 high performance liquid chromatography system

using a SuperdexTM 200 10/300 GL (10 mm × 300 mm; GE Healthcare) size exclusion column with a

13

mobile phase of PBS buffer (pH 7.4) at a flow rate 0.5 mL/min, as described previously (9). All

antibodies were analyzed at a concentration of 0.5 mg/mL in a sample volume of 100 μL.

Chromatograms were obtained by monitoring the absorbance at 280 nm. The apparent molecular

mass of samples was estimated by fitting the elution volume into the calibration curve obtained from

running of molecular mass standard markers (alcohol dehydrogenase, 150 kDa; bovine serum albumin,

66 kDa; carbonic anhydrase, 29 kDa) (Sigma-Aldrich).

Far-UV CD spectroscopy

Far-UV CD spectra (195-260 nm) of the purified Fc proteins (0.1 mg/mL in PBS buffer, pH 7.4) were

analyzed on a Chirascan plus (AppliedPhotophysics, UK) at 25°C, in a 0.05 cm path length quartz

cuvette with a step size of 1 nm. Averaged spectra of three scans were corrected for buffer blank

(PBS buffer, pH 7.4) and expressed as mean residue ellipticity [Θ] (deg∙cm2∙dmol-1) (12).

Differential scanning calorimetry (DSC)

DSC was performed using a MicroCal VP-DSC Microcalorimeter (GE Healthcare). Fc proteins (2.5

mg/mL (~50 μM) in 10 mM Tris, 10 mM NaCl buffer (pH 8.0)) were heated from 25°C to 95°C at a

heating rate of 1.5°C/min. After subtraction of the buffer reference scan, sample thermogram was

fitted into a two-state transition model to capture the melting temperatures (Tm) of CH2 and CH3

domains in the Origin 7.0 graphing software supplied by MicroCal.

SPR analysis

Kinetic binding interactions of antibodies or Fc proteins to target proteins (Met-Fc, VEGFR-2-Fc,

FcγRs, and FcRn) were determined at 25°C by a Biacore 2000 SPR biosensor (GE Healthcare), as

described before (13). The target proteins at a concentration of 20 μg/mL in sodium-acetate buffer

(pH 4.0) was immobilized onto the CM5 chip at a level of about 1,000 response units (RUs). Before

loading the analytes, the chip was equilibrated with HBS-EP buffer (10 mM HEPES, 150 mM NaCl,

14

0.005% v/v surfactant P20, pH 7.4). Antibodies or Fc proteins in HBS-EP buffer was injected into

flow cell at a flow rate of 30 μL/min for 3 min, which was followed by the dissociation phase by

injection of HBS-EP buffer at 30 μL/min for 3 min. Between cycles, flow cell were regenerated

with 1 M NaCl, 20 mM NaOH buffer (Met-Fc, VEGFR-2-Fc binding assay), 100 mM NaCl, 2 mM

NaOH buffer (FcγRs binding assay), or pH 8.0 HBS-EP buffer (FcRn binding assay) at 30 μL/min for

1 min. Association (kon) and dissociation (koff) rate constants were determined by the 1:1 binding

model using the BIAevaluation software (13).

In the assay monitoring the simultaneous binding interactions, bsVeMet (100 nM) was first

flowed over VEGFR-2-Fc-immobilized surface (~1000 RU) at 30 μL/min for 3 min and then, without

the dissociation phase, a second protein (Met-Fc or VEGFR-2-Fc at 100 nM) in HBS-EP buffer was

immediately injected at 30 μL/min for 3 min (13).

Sandwich ELISA

The indicated antibodies (3 μM) were first incubated for 1 h with plate-coated VEGR2-Fc (1

μg/well) in 96-well plates (SPL, Korea). After washing with PBST (PBS plus 0.1% Tween-20),

serially diluted 6×His-tagged human Met-Fc (0.16 – 100 nM) was added and incubated at room

temperature for 1 h. Bound Met-Fc protein was detected using mouse anti-His tag antibody (IG

Therapy, Korea) and alkaline phosphatase conjugated goat anti-mouse antibody with the substrate of

p-nitrophenyl phosphate (Sigma-Aldrich) (13). Absorbance at 405 nm was read with a VersaMax

microplate reader (Molecular Devices, Crawley, UK).

Flow cytometry

Cell surface expression levels of Met and VEGFR-2 were determined by flow cytometry after indirect

immunofluorescent labeling using mouse anti-Met (Cell Signaling) and anti-VEGFR-2 antibody

(Abcam), respectively, followed by FITC-conjugated anti-mouse IgG (PIERCE) (14). Binding of

msMet, tanibirumab, and bsVeMet antibodies to HUVEC and MKN45 cells were determined by

15

labeling cells with 100 nM antibodies for 1 h and then secondary labeling with FITC-conjugated anti-

human IgG Fc antibody (Sigma) before flow cytometric analysis.

Cell proliferation assay

HUVECs, seeded at a density of 0.4 × 104 cells/well in 96-well plates, were incubated at 37°C in

complete media for 24 h. Then, cells were starved using EBM-2 medium for 4 h prior to treatments

of HGF (50 ng/mL), VEGF (20 ng/mL), and/or the antibodies, as specified in the figure legend.

MKN45 cells, seeded at a density of 0.6 ×104 cells/well in 96-well plates, were incubated at 37 °C in

complete media for 24 h. Then, cells were serum-starved using free FBS RPMI1640 for 6 h before

treatments of HGF (50 ng/mL) and/or the antibodies, as specified in the figure legend. Cell viability

was analyzed using the MTT assay kit (Sigma-Aldrich) and the results are presented as the percentage

of viable cells versus the medium-treated control or the corresponding control (12, 14).

Western blots

HUVECs were cultured for 6 h in serum-free medium, then pretreated with antibodies (0.3 μM) at

37°C for 1 h, and then with or without HGF (10 or 20 ng/mL) and/or VEGF (10 or 20 ng/mL) at the

indicated concentrations for 10 min (15). MKN45 cells were cultured overnight in serum-free

medium, then pretreated with antibodies (0.3 μM) at 37°C for 2 h, and then treated with or without

HGF (20 ng/mL) for 10 min. Whole cell lysates were extracted using RIPA buffer (Biosesang), and

protein concentrations were determined using Bicinchoninic Acid (BCA) Kit (Sigma). Western

blotting for cell lysates treated as specified in figure legends was performed following the standard

procedure (14). Proteins were visualized using a PowerOpti-ECL western blotting detection reagent

(Animal Genetics, Inc. Korea) and an ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ)

(16). Equal amount of cell lysates were analyzed by western blotting with β-actin as a loading

control.

16

Animal experiments

All animal experiments were evaluated and approved by the Animal and Ethics Review

Committee of Ajou University and performed according to the guidelines established by the

Institutional Animal Care and Use Committee. Female BALB/c athymic nude mice, aged 4 weeks

and weighed between 15-20 g, were purchase from NARA bio (Korea). MKN45 cells (5 × 106 cells

per mouse) were inoculated subcutaneously in the right thigh of nude mice. When the mean tumor

size reached about ~100 mm3 (after 7 day growth), mice were randomized into each groups (n = 8 per

group) and administered intravenously via the tail vein with antibodies at the equivalent molar amount

(200 μL of 6.25 μM per injection) as specified in the figure legends. Control mice were given equal

volume of PBS. The tumor size and body weight were measured thrice per week using calipers, and

tumor volumes were calculated by the formula ½ × length × width2 (12). Animals were sacrificed if

tumor size exceeded 2,000 mm3. The statistical significance was evaluated by one-way analysis

followed by two-tailed student t tests on Excel Software (Microsoft Inc.).

Immunofluorescence of tumor tissues

A single, equivalent molar dose of antibodies (200 μL of 3.1 μM per injection) was intravenously

injected into mice carrying MKN45 xenografted tumors with an average size of ~7-8 mm in diameter.

Tumor tissues were harvested 5 or 24 h postinjection. Tumor preparation and staining of the

cryosections were performed as described before (17, 18). Briefly, the excised tumors from mice

were fixed in 4% Paraformaldehyde (PFA) overnight at 4°C, cryo-protected in 30% sucrose for 10 h

and then frozen in Tissue-Tek OCT (Optimal Cutting Temperature) embedding medium. For

immunofluorescence staining, cryosections were prepared at 10 μm thickness and incubated with

blocking solution (2% BSA in PBS) for 1 h at 25°C. Tissue sections were stained with rat anti-

mouse CD31 mAb (BD Biosciences) at 4°C overnight, washed 2× with PBST (PBS containing 0.1%

Tween20) and then stained with goat anti-rat TRITC-conjugated antibodies (Millipore) and FITC-

conjugated anti-human IgG Fc antibody (Sigma) at 25°C for 1.5 h. Slides were then washed 3× with

17

PBST and then mounted in Vectashield (mounting medium with DAPI, Vector Laboratories,

Burlingame, CA). Tissue sections were examined by Ziess LSM710 systems with ZEN software

(Carl Zeiss). Fluorescence in each tissue was quantified with Image J (National Institutes of Health,

USA).

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