Supplementary Materials for

Elucidating the interplay between IgG-Fc valency and FcγR activation

for the design of immune complex inhibitors

Daniel F. Ortiz, Jonathan C. Lansing, Laura Rutitzky, Elma Kurtagic,

Thomas Prod'homme, Amit Choudhury, Nathaniel Washburn, Naveen Bhatnagar,

Christopher Beneduce, Kimberly Holte, Robert Prenovitz, Matthew Child,

Jason Killough, Steven Tyler, Julia Brown, Stephanie Nguyen, Inessa Schwab,

Maurice Hains, Robin Meccariello, Lynn Markowitz, Jing Wang, Radouane Zouaoui,

Allison Simpson, Birgit Schultes, Ishan Capila, Leona Ling, Falk Nimmerjahn,

Anthony M. Manning, Carlos J. Bosques*

*Corresponding author. Email: cbosques@momentapharma.com

Published 16 November 2016, Sci. Transl. Med. 8, 365ra158 (2016)

DOI: 10.1126/scitranslmed.aaf9418

The PDF file includes:

Materials and Methods

Fig. S1. Generation and characterization of FcM-related materials

Fig. S2. Characterization of multivalent Fc constructs.

Fig. S3. Monocyte activation dose response of Fc constructs.

Fig. S4. Analytical characterization of Fc3Y.

Fig. S5. Binding of IgG1, a trimeric IgG1 IC, Fc, and Fc3Y to FcγRs.

Fig. S6. Comparison of Fc3Y binding to platelets and other FcγR-expressing

cells.

Fig. S7. Lack of induction of FcγRIIb phosphorylation by Fc3Y in Daudi B cells.

Fig. S8. Lack of neutrophil activation by Fc3Y.

Fig. S9. Fc3Y binding to Raji cells and lack of induction of ADCC.

Fig. S10. Fc3Y inhibition of moDC activation.

Fig. S11. Role of FcγRs, B cells, and endogenous IgG in an ITP mouse model.

Fig. S12. Fc3Y inhibition of FcγRIV-dependent 6A6-IgG2a–mediated platelet

depletion in an ITP mouse model.

Fig. S13. Pharmacokinetic analysis of Fc3Y.

Table S1. Relative binding of Fc oligomers to FcγRs.

Legend for table S2

Reference (57)

www.sciencetranslationalmedicine.org/cgi/content/full/8/365/365ra158/DC1

Other Supplementary Material for this manuscript includes the following:

(available at

www.sciencetranslationalmedicine.org/cgi/content/full/8/365/365ra158/DC1)

Table S2. Source data (provided as an Excel file).

Materials and Methods

FcM purification and fractionation

Protein expressed transiently in HEK293 cells was captured on protein A (Poros MabCapture A,

ThermoFisher Scientific) and fractioned by strong cation exchange (Poros XS, ThermoFisher

Scientific) with a step gradient from 0% B to 40% B (buffer A: 50 mM MES, pH 6.4; buffer B:

10 mM MES, 500 mM sodium chloride, pH 6.4). At 40% B buffer composition, the gradient was

held for 1.5 CV and then increased to 100% B in 5.5 CV. Components eluting after the 40% B

hold were combined to produce FcM. FcM was further fractionated by SEC (tandem Hi Prep S-

300 HR and S-400 HR Sephacryl columns, GE Healthcare Life Sciences) into four fractions (see

fig. S1A).

Immune complex formation

ICs were formed and purified as described (57). Briefly, plasma from SLE patients (All Cells)

was pooled (10 patients), treated with 500 µg/mL of HeLa nuclear extract (Santa Cruz

Biotechnology) for 1 h, mixed 1:1 with 7% polyethylene glycol (PEG) 8000 (Sigma-Aldrich),

and incubated overnight at 4°C. Precipitated SLE-ICs were pelleted by centrifugation at 14,000

rpm for 10 min, washed in 3.5% PEG, and resuspended in PBS.

Goat IgG:rabbit anti-goat IgG ICs were generated by mixing equal volumes of goat IgG and

rabbit anti-goat IgG, each at 1.8 mg/mL concentration, and incubating at 37°C for 30–60 min.

ICs were held for 0–60 min on ice before testing.

Fc-oligomer production

Genes were subcloned into the pcDNA3.4 A_357 expression vector (ThermoFisher Scientific),

and plasmids were transfected into EXPI293 cells (ThermoFisher Scientific) according to the

manufacturer’s instructions. Fc-oligomers were purified by protein A capture (POROS

MabCapture A, ThermoFisher Scientific), followed by cation exchange (Poros XS,

ThermoFisher Scientific).

Mice

Female C57BL/6 mice weighing 18-22 g were obtained from Charles River Laboratories for ITP

studies, male C57BL/6 mice weighing 22–30 g and 8–12 weeks of age were obtained from

Jackson Laboratories for CAIA studies, and age-matched C57BL/6 mice 8-16 weeks of age were

obtained from Janvier for EBA studies. Mice for ITP and CAIA animal studies were used after a

minimum of 1 week acclimation, and studies were conducted in compliance with the National

Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals under protocol

numbers 12-0809-1 and 14-1218-1 and approved by the Institutional Animal Care and Use

Committee of Momenta Pharmaceuticals, Inc. Mice for EBA experiments were maintained under

specific pathogen-free conditions, and studies were performed with the approval of the

Government of Lower Franconia in Germany (study number 55.2-2532.2-116) and according to

the guidelines of the NIH and the legal requirements in Germany and the USA.

Non-reducing SDS-PAGE

Samples were denatured in sample buffer (4% SDS, Bio-Rad) at 65 °C for 10 min and run on a

Criterion TGX stain-free gel (4-15% polyacrylamide, Bio-Rad). Gels were imaged by ChemiDoc

MP Imaging System (Bio-Rad).

Analytical size exclusion chromatography (SEC)

Samples were analyzed at 30 °C on an Agilent 1200 system using a Zenix-C 4.6 × 300 mm 3 m

particle size column (Sepax Technologies) at an isocratic flow of 0.35 mL/min with 150 mM

sodium phosphate pH 7.0.

Analytical ultracentrifugation

Analytical ultracentrifugation (AUC) was performed on a ProteomeLab XL-1 (Beckman

Coulter) at a rotor speed of 42,000 rpm for 8 h. Data were fit using a continuous sedimentation

coefficient model. The partial specific volume was set to 0.73, F-ratio to 0.68, frictional

coefficient to 1.2, and SMax to 60. The density and viscosity of the buffer were 0.99823 g/mL and

0.01018 P, respectively.

Valency determination by surface plasmon resonance

Binding experiments were performed on a Biacore T200 instrument (GE Healthcare) using a

CM3 Series S sensor chip. Native Protein A was immobilized via direct amine coupling. Ligands

were diluted in running buffer and captured. A 6-point dilution series of human recombinant

CD32a (R&D Systems) was prepared and flowed over the captured ligands. After double-

referencing, equilibrium analysis using the steady-state model was performed on each set of

curves. The valency of each ligand was calculated using (1).

Ligand Valency = Rmax/[(MW analyte/MW ligand)*Ligand Capture Level] (1)

Fc3Y and IVIg binding to blood cells

Whole blood was pre-incubated for 45 min at 37 °C with VivoTag645 labeled Fc3Y or IVIg

diluted 2X in 100 µl of complete medium (RPMI 1640/10% FBS/ 0.05 mM 2-mercaptoethanol/2

mM glutamine). A 1:2 serial dilution starting at 1,600 µg/mL was set up to generate binding

curves. Blood was then lysed with ammonium chloride, and cells were washed with PBS and

stained with the Fixable Viability Dye eFluor 506 (eBioscience). Cells were washed again and

stained for 25 min on ice with an antibody cocktail to phenotype granulocytes (PerCPCy5.5 anti-

CD66b), monocytes (APC/Cy7 anti-CD14), dendritic cells (PE anti-CD11c), NK cells and NKT

cells (PE/Cy7 anti-CD56), B cells (AF488 anti-CD19), and T cells (Pacific Blue anti-CD3). In

some experiments, leukocytes obtained from lysed whole blood (500,000 cells/condition) or

platelets present in whole blood (20 µL/condition) were incubated with VivoTag 645-labeled

Fc3Y at 1.6, 16, 160, and 1,600 µg/mL for 15 min at 4 °C and RT, respectively. After Fc3Y

binding, samples were stained for additional 30 min with specific markers to phenotype the

different cells. The Ab cocktail used to characterize the different leukocytes was composed of

FITC anti-CD11c, PerCPCy5.5 anti-CD19, PE-Cy7 anti-CD56, APC-Cy7 anti-CD14, and

Pacific Blue anti-CD3. With this mAb cocktail, granulocytes were identified based on their SSC

and FSC characteristics. The Ab cocktail used to characterize platelets was composed of FITC

anti-CD32, PE anti-CD62P, PE-Cy7 anti-CD45, and Pacific Blue anti-CD41 (all mAb from

BioLegend). After staining with mAbs, leukocytes were washed with FACS buffer (1X PBS, 1%

BSA, 0.05% NaN3) once and resuspended in 200 µL of 1% paraformaldehyde. Whole blood was

diluted to 2 mL with a buffer containing 10 mM HEPES, 140 mM NaCl, 2.7 mM KCl, 2 mM

MgCl, 5.5 mM glucose. Samples were acquired in a FACSVerse instrument (BD Biosciences).

For acquisition of platelets in diluted whole blood, a platelet gate was set at low forward light

scatter (FSC) profile to discriminate platelets from red blood cells and leukocytes. Data were

analyzed with FlowJo software (Version 7.6.5, Tree Star).

Granulocytes (SSChigh/live/singlet/CD66b+), monocytes (SSClow/live/singlet/CD11c±,CD14+),

dendritic cells (SSClow/live/singlet/CD11c+,CD14-), NK cells (SSClow/live/singlet/CD11c-,CD14-

/CD3-, CD56+), NKT cells (SSClow /live/singlet/CD11c-, CD14-/CD3+, CD56+), T cells

(SSClow/live/singlet/CD11c-, CD14-/CD3+, CD56-), and B cells (SSClow/live/singlet/CD11c-,

CD14-/CD3-, CD56-/CD19+). Binding data are expressed as the geometric mean of the

fluorescence intensity (MFI) of the VivoTag645 signal (APC-channel).

IL-6 release in PBMCs

FcR-mediated cytokine production in PBMCs was stimulated by plate-bound IgG1 as described

for IL-8 release in monocytes. PBMCs were isolated from whole blood using Histopaque

(Sigma-Aldrich) and resuspended at 1.25 × 106 cells/mL in complete medium. PBMCs were

added in 100 L to the IgG1- and PBS-coated wells. Cells were incubated for 24 h before the

removal of the culture supernatant for analysis of IL-6 production by Luminex (Millipore).

Calcium flux in neutrophils

Neutrophils were isolated from whole blood lysed with ammonium chloride by negative

selection (EasySep Human Neutrophil Enrichment Kit, StemCell Technologies). Purity of

neutrophils was > 95% as assessed by FACS. Cells were resuspended in RPMI/2% BSA medium

and incubated in Fura-2 Calcium Dye (Molecular Devices) for 45 min at 37°C. Dyed neutrophils

were read on the FlexStation plate reader. Fc3Y and IVIg were added in a dose response curve

17 s after start of reading. ICs derived from SLE plasma were added 90 s after the start and read

for an additional 110 s. Calcium flux is reported as the ratio between bound and unbound

calcium (340/380 nm).

Antibody-dependent cell-mediated cytotoxicity

PBMCs were isolated from buffy-coats (Research Blood Components) or whole blood by Ficoll

gradient centrifugation (GE Healthcare Life Sciences). Cells were either used fresh or cultured

overnight in X-Vivo medium. PBMCs were then plated with Raji cells at a ratio of 24:1 effector

(PBMC): target (Raji) in X-Vivo medium. Medium, rituximab (at 2 µg/mL or 10 μg/mL), Fc3Y,

or IVIg were added simultaneously, and the plates were incubated 4 h at 37 °C. Culture

supernatants were tested for lactate dehydrogenase concentrations as a measure of cell death

using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega).

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) assay

Samples at 1 mg/mL were mixed with the denaturing buffer supplied in the HT Protein Express

Reagent kit (PerkinElmer). The mixture was heated at 40°C for 20 min. Samples were

transferred to a 96-well plate after adding 70 L of water and then injected into a Caliper GXII

instrument (PerkinElmer) equipped with the HT Protein Express LabChip (PerkinElmer). The

relative abundance of each size variant was calculated based on fluorescence intensity.

SEC coupled to mass spectrometry

Fc3Y or FcM were diluted to 2 μg/μL in running buffer (78.98% water, 20% acetonitrile, 1%

formic acid [FA], and 0.02% trifluoroacetic acid). SEC separation was performed on two Zenix-

C SEC-300 columns (2.1 × 350 mm; Sepax Technologies), total length of 700 mm, using a

mobile phase consisting of 78.98% water, 20% acetonitrile, 1% FA, and 0.02% trifluoroacetic

acid at a flow rate of 80 μL/min. Mass spectra were acquired on a QSTAR Elite (AB Sciex) Q-

ToF mass spectrometer operated in positive mode. The neutral masses under the individual size

fractions were deconvoluted using Bayesian peak deconvolution by summing the spectra across

the entire width of the chromatographic peak.

Glycosylation analyses by glycopeptide liquid chromatography–mass spectrometry

Fc3Y was reduced with dithiothreitol and alkylated with iodoacetamide in 6 M guanidine HCl in

100 mM ammonium bicarbonate pH 7.5. Digestion with trypsin was carried out in a Barocycler

(Pressure Biosciences) for 1 h at 37°C, cycling between 20,000 PSI and ambient pressure 30

times. Peptides were separated on a Waters BEH PepMap C18 column (1 × 100 mm, 1.7 μm

particle size). N-glycopeptide relative abundance was determined based on the integrated area of

the extracted ion current for the z=2 and z=3 ion for each species.

Preparation of TNFα/anti-TNFα Complexes.

Recombinant, monoclonal anti-TNFα antibody (prepared in house) and soluble, trimeric TNFα

(R&D Systems) were mixed in a 3:1 molar ratio to form complexes composed of three anti-

TNFα molecules binding one TNFα trimer. Mixtures were incubated for 24 hours at 37˚C. The

average molecular weights of TNFα immune complexes were measured using an SEC system

coupled with Multi-Angle Light Scattering (MALS) and Refractive Index (RI) detection. The

SEC column used was an Agilent Bio SEC-5, at 1000 Å, 4.6 x 300 mm, 5 µm pore size. The

light scattering and refractive index systems were Wyatt mini DAWN Multi-Angle Scattering

Detector and Wyatt Optilab rEX Refractive Index system, respectively. The chromatographic

system, MALS, and RI were equilibrated at 0.35 mL/min with 150 mM sodium phosphate

solution, pH 7.0. A dn/dc (the rate of change of the refractive index with the concentration of a

solution for a sample at a given temperature, a given wavelength, and in a given solvent) of

0.185 mL/g was used to calculate the average molecular weight of each species. Each sample

(monomer or complex) were analyzed directly after sample preparation.

Cell-based competitive FcR binding

Relative binding of Fc-oligomers and controls to FcRs was measured using cell-based

homogeneous time-resolved fluorescence competition assays (TR-FRET CisBio assays). Kits

used were CD16aV158, CD32aH131, CD32b, and CD64. Assay reagents were prepared

according to the manufacturer’s instructions. A 10-point, 3-fold serial dilution series, plus one

blank per sample, was generated using an automated liquid handler (Freedom EVOware 150,

Tecan). Assay plates were read on a PHERAstar fluorescent reader (BMG Labtech GmbH) at

665 and 620 nm.

Pharmacokinetic study

Female C57BL/6 mice (n=12, 8-10 weeks old), were dosed i.v. with 0.1 g/kg of Fc3Y. Blood

samples (50 μL) were collected from cheek bleeds of four mice per time point at alternating

times 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 1 day, 2 days, 3 days, 4 days, and 5 days. All mice

were bled at 7, 9, 11, 14, 16, 21, and 23 days. Fc3Y serum concentrations were determined by

ELISA.

Fig. S1. Generation and characterization of FcM-related materials. (A) Preparative SEC

fractionation of FcM on tandem S-400HR and S-300HR resin. (B) Analytical SEC analyses of

FcM-related samples on tandem S-400HR and S-300HR resin confirm the size distribution

observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Fig. S2. Characterization of multivalent Fc constructs. (A) Molecular weight distribution

determination by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of products

generated from uncontrolled multimerization of tandem wild-type Fc domains. (B) Capillary

electrophoresis-sodium dodecyl sulfate analysis of discrete Fc-polymeric structures to determine

size distributions. (C) SEC analyses of Fc oligomers assess size distribution and non-covalent

aggregation.

Fig. S3. Monocyte activation dose response of Fc constructs. (A) SYK phosphorylation in THP-1 cells. (B) Calcium flux in

primary monocytes (mean ± SD from technical replicates, n = 3). p/t, phosphorylated/total; RFU, relative fluorescence unit.

Fig. S4. Analytical characterization of Fc3Y. (A) Fc3Y has a simple glycan distribution with

little additional posttranslational modification, as demonstrated by liquid chromatography–mass

spectrometry (LC-MS). (B) Fc3Y N-glycosylation as determined by glycopeptide LC-MS. cps,

counts per second; XIC, extracted-ion chromatogram.

Fig. S5. Binding of IgG1, a trimeric IgG1 IC, Fc, and Fc3Y to FcγRs. (A) TNF/anti-TNF

complexes (Anti-TNF:TNF IC) were mixed in a 3:1 molar ratio, incubated at 37 ˚C for 24 hours,

and then analyzed by SEC-MALS. Molecular weights were determined and overlaid with an

anti-TNF antibody standard. (B) Comparison of anti-TNF and anti-TNF:TNF IC binding to

FcRs. EC50s were measured for binding on cells expressing FcγRIIa-H131, FcγRIIb, and

FcγRIIIa-V158 variant in a TR-FRET competition assay. (C) Comparison of Fc1 and Fc3Y

binding to FcRs. EC50s were measured for binding on cells expressing FcγRIIa-H131, FcγRIIb,

and FcγRIIIa-V158 in a TR-FRET competition assay.

Fig. S6. Comparison of Fc3Y binding to platelets and other FcγR-expressing cells. VivoTag

645-labeled Fc3Y was incubated with either leukocytes obtained from lysed whole blood or

platelets present in whole blood for 45 min at 4 °C and RT, respectively. After the initial 15 min-

incubation with Fc3Y, samples were stained with specific markers to phenotype the different

cells. Flow cytometry results are expressed as geometric mean fluorescence intensity (MFI) and

are from one experiment representative of five. Analysis of T cells was included as a negative

control for binding because they do not commonly express FcRs.

F c 3 Y c o n c e n tra t io n (µ g /m L )

MF

I

01.6 1

6160

1600

0

3 0 0 0

6 0 0 0

9 0 0 0

1 2 0 0 0

G ra n u lo c y te s

M o n o c y te s

D C s

N K c e lls

B c e lls

N K T c e lls

T c e lls

P la te le ts

Fig. S7. Lack of induction of FcγRIIb phosphorylation by Fc3Y in Daudi B cells.

Immunoblots of extracts from Daudi B cells (3 x 106 cell/mL) which had been incubated for 30

min at 37 °C with vehicle (C), 5 µg/ml rituximab (RTX, positive control), or Fc3Y (10, 50, or

100 μg/mL). Cell extracts were separated by SDS-PAGE, and immunoblots were stained for

phospho FcγRIIb and beta actin.

Fig. S8. Lack of neutrophil activation by Fc3Y. Human neutrophils were stained with Fura-2

calcium dye before being incubated with IVIg, Fc3Y, and ICs isolated from the plasma of

patients with systemic lupus erythematosus (SLE-IC; 750 μg/mL). Calcium flux was measured

on a FlexStation plate reader. Technical replicates are shown (n = 2).

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

1 .0 1 .5 2 .0 2 .5 3 .0

L o g (µ g /m L )

Ca

lciu

m F

lux

(34

0/3

80

Ra

tio

ma

x-m

in)

F c3Y

IV Ig

S L E -IC

     

Fig. S9. Fc3Y binding to Raji cells and lack of induction of ADCC. (A) Raji cells were

stained with 160 µg/mL of VivoTag labeled Fc3Y for 25 min at 4 °C in the dark. After staining,

cells were washed twice and analyzed by flow cytometry. Results are expressed as percentage of

Fc3Y+ cells as compared to the unstained buffer control. (B) PBMC and Raji cells were

incubated with the indicated concentrations of Fc3Y or 10 µg/mL of rituximab (positive control)

for 4 h at 37 °C. Culture supernatants were tested for LDH concentration. The extent of

rituximab-induced ADCC of Raji cells was considered 100% ADCC. Values are reported after

subtracting the background cell death from cultures with PBMC and Raji cells alone. Mean ± SD

is shown for technical replicates (n=3).

Fig. S10. Fc3Y inhibition of moDC activation. Monocyte-derived dendritic cells (moDCs)

were incubated with medium (CTRL, black histogram), IVIg (green histogram), and Fc3Y (blue

histogram) before stimulation with plate-bound IgG1 (solid gray histogram). Surface expression

of the costimulatory molecule CD86 was measured by flow cytometry. Fc3Y inhibited the

activation of moDCs by plate-bound IgG as determined by the reduction in CD86 surface

expression. Horizontal lines within the graphs indicate the gate used to identify activated

dendritic cells. Results are representative of 6 independent experiments.

Fig. S11. Role of FcγRs, B cells, and endogenous IgG in an ITP mouse model. (A) IgG1 anti-

CD41 depletion of platelets requires FcRIII. Control C57BL/6 mice and animals lacking the

FcR common chain, FcRI, FcRIIb, FcRIII, or FcRIV were treated with anti-CD41 IgG1

antibody to induce platelet depletion. FcRIII deficient mice did not exhibit significant platelet

depletion. Prophylactic treatment with Fc3Y at 0.1 g/kg effectively inhibited platelet reduction in

control mice. P values indicate statistically significant difference from the C57BL/6 group. (B)

Fc3Y prevention of platelet depletion does not require B cells or endogenous Igs. Control

C57BL/6 and Rag2-c deficient mice, which do not develop mature B cells or T cells, were

treated with anti-CD41 antibody. Prophylactic treatment with Fc3Y at 0.1 g/kg effectively

prevented platelet depletion in both control and Rag2-c deficient mice.

Fig. S12. Fc3Y inhibition of FcγRIV-dependent 6A6-IgG2a–mediated platelet depletion in

an ITP mouse model. (A) Control C57BL/6 mice and animals lacking the FcR common chain,

FcRI, FcRIIb, FcRIII, or FcRIV were treated with anti-platelet 6A6-IgG2a antibody. FcRIV

deficient mice did not exhibit significant platelet depletion. P values indicate statistically

significant difference from the C57BL/6 group. (B) Prophylactic treatment with Fc3Y at 0.1 g/kg

effectively inhibited 6A6-IgG2a mediated platelet reduction in control mice.

Fig. S13. Pharmacokinetic analysis of Fc3Y. Female C57BL/6 mice (8-10 weeks old, n=12)

were dosed i.v. with 0.1 g/kg of Fc3Y. Fc3Y serum concentrations were measured at different

time points by ELISA. Mean ± SEM (n=4-12) are shown for biological replicates.

Sample FcγRI FcγRIIa FcγRIIb FcγRIIIa

IVIG 0.30 52 24 7

Fc2 0.34 558 17 74

Fc3L 1.05 6042 122 119

Fc3Y 2.46 6304 1483 1497

Fc5X 3.59 13254 4224 3722

Fc5Y 1.43 11472 994 6291

Table S1. Relative binding of Fc oligomers to FcγRs. Avid binding was measured using the

TR-FRET competitive binding assay. Data are presented as fold change compared to binding of

Fc1. Values were determined as mean EC50 for two experimental replicates, each consisting of 2

technical replicates.