Supplementary Methods:

Recombinant Proteins and Inhibitors

Recombinant ADAMTS-4 and ADAMTS-5 (262-624) were generated as described [1]. ADAMTS-5

(262-484) was expressed as ADAMTS-5 (1-484) with C-terminal Strep-Tag in a Chinese hamster ovary

(CHO) cell line and purified from the conditioned medium via Strep-Tactin resin. ADAMTS-5 (1-567)

and ADAMTS-5 (1-930) were expressed with C-terminal affinity tags in CHO cell lines; the prodomains

were naturally processed during expression to generate ADAMTS-5 (262-567) and ADAMTS-5 (262-

930). Due to endogenous proteolytic processing of the C-terminal affinity tags, conventional

chromatography schemes were developed for purification. For ADAMTS-5 (262-567) the conditioned

medium was concentrated and filtered prior to ammonium sulfate precipitation (20/55% saturations); the

solubilized ammonium sulfate pellet was chromatographed on Source 15 Phenyl followed by sizing on

Superdex 200. ADAMTS-5 (262-930) was captured from conditioned medium by Source 15 Q followed

by Heparin Sepharose. The Heparin Sepharose eluate was precipitated with 45% ammonium sulfate

saturation followed by sizing on Superdex 200. Appropriate N-terminal sequence was confirmed by

Edman sequencing. Activity of purified ADAMTS-4 and ADAMTS-5 proteins was confirmed using a

synthetic aggrecan IGD peptide substrate assay [1]. ADAMTS-5 and ADAMTS-4 selective mAbs (Table

1) and isotype matched control mAbs were expressed and purified from culture media via affinity

chromatography from hybridomas (murine forms) or stably transfected Chinese hamster ovary (CHO)

cell lines (humanized forms). Recombinant MMPs, aggrecan IGD (R&D Systems) and purified bovine

aggrecan (Sigma Chemical) were purchased. GSK571949 is a broad spectrum small molecule

metalloprotease inhibitor [1] used as an assay control in some experiments.

Immunization, mAb selection and humanization

SJL mice (Charles River), immunized with a combination of purified recombinant human ADAMTS-4

and ADAMTS-5, were routinely monitored for specific serum immunoreactivity and neutralization of

specific protease activity. Splenocytes and lymph node-derived cells from mice with desirable profiles

were progressed to fusion with P3XBcl-2.13 mouse myeloma cells and cultured under hypoxanthine-

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aminopterin-thymidine (HAT) selection. Resulting hybridomas were screened for binding specificity and

inhibition of aggrecanase activity (independently against a purified bovine aggrecan and synthetic human

peptide [QTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLK–biotin]) substrate to identify

hybridomas of interest and cloned by limiting dilution. Humanization of murine antibodies was

performed essentially as described [2] using a best-fit CDR grafting technique. Briefly, mouse CDRs

were grafted onto suitable human acceptor frameworks which showed high sequence homology with the

original framework region of each mAb. GSK-proprietary databases of human germline sequences were

searched to identify best match frameworks suitable for grafting of the CDRs. Key residues in the mouse

framework were identified which might be important in maintaining the correct conformation of the

CDRs. These were considered as possible “back mutations” which might be necessary to retain the

binding affinity and were incorporated into the various designs for the humanized molecules. Humanized

variants were selected by retained/increased binding affinity/selectivity and functional potency

ARGS Neoeptiope detection

Assay plates were coated with a monoclonal anti-aggrecan capture antibody (Invitrogen, Cat# AHP0022),

washed and experimental samples were added to the wells followed by 2hr incubation. Plates were

washed, and incubated with a biotin (DELFIA) or SulfaTag (electrochemiluminescent) labeled ARGS

neoepitope specific detection antibody [3] (OA-1, 50ng/well) followed by another wash. Captured ARGS

neoepitope was detected using streptavidin−europium (500pg/well) for 30 min and enhancement solution

(200μL/well) for 5 min followed by europium signal acquisition (320 nm excitation, 615 nm emission) on

an Envision plate reader (DELFIA) or directly in a MSD SECTOR Imager 6000

(electrochemiluminescent). In the case of serum analysis ARGS concentrations were calculated using a

standard curve of ADAMTS-5-treated human aggrecan IGD as described [4].

Substrate selectivity

GSK2394002 was assessed for the ability to inhibit ADAMTS-5-mediated proteolysis of two substrates

(aggrecan and brevican) using an in vitro biochemical assay format. Incubation of purified human

ADAMTS-5 [100nM final] with varying concentrations of GSK2394002 [ranging from 15.6 – 1000nM

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final] was performed in individual tubes for 30 minutes to allow for binding. Following pre-incubation,

2ug of recombinant human aggrecan IGD [1.4uM final] or brevican [1.02uM final] substrate (both from

R&D Systems) was added and incubated for 1 hour. Separate tubes containing: ADAMTS-5 alone,

aggrecan IGD alone, aggrecan IGD + ADAMTS-5, GSK2394002 alone, GSK2394002 + ADAMTS-5,

brevican alone and brevican + ADAMTS-5 were run in parallel as controls. All components were diluted

in assay buffer (10mM HEPES, 150mM NaCl2, 1uM ZnCl2, 1mM CaCl2, 0.05% NP-40 at pH 7.4) and

reactions were performed at 37oC in 25uL total volume. The reactions were stopped with addition of 2uL

of 270uM EDTA [20uM final]. Differential substrate proteolysis in each sample was resolved by

electrophoresis on NuPAGE Novex 4-12% Bis-Tris gradient gels (Invitrogen) with MOPS running buffer.

Gels were stained with SimplyBLUE total protein stain (Invitrogen) and images captured using a Licor

Odyssey imaging system.

Crystallography

Mouse anti-human ADAMTS-5 mAbs 7B4.1E11 and 12F4.1H7 were expressed in mouse

hybridoma cultures and purified. Fab fragments were generated by papain cleavage using an

ImmunoPure Fab Preparation Kit (Pierce) and used without additional purification. Fabs were

concentrated and crystallized using the hanging drop vapor diffusion method. Structures were solved by

X-ray diffraction and submitted to Protein Data Bank (PDB); 7B4.1E11 Fab at 2.6Å resolution (PDB:

4X80) and 12F4.1H7 Fab at 2.3Å resolution (PDB:4X8J). The ADAMTS proteins used for subsequent

modeling were publically available and obtained directly from the PDB site: ADAMTS-4 catalytic +

disintegrin (apo form) 2.8Å resolution (PBD: 3B2Z) and ADAMTS-5 catalytic + disintegrin with

inhibitor bound 2.6Å resolution (PDB: 2RJQ).

Structure modeling

Computational mAb Structural Humanization

In order to generate humanized structures of the ADAMTS-5 Fabs (GSK2394000 and GSK2394002) and

the murine ADAMTS-4 Fab 7E8.1E3, the protein sequences (amino acid sequences listed below) in

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Molecular Operating Environment (MOE) antibody modeler [5,6]. The MOE modeler employs a

knowledge-based approach that utilizes a database of available antibody structures (available in PSILO

database; Chemical Computing Group, Montreal). In Fab homology modeling, appropriate templates for

the framework region and CDR loops were first found by searching against the antibody structure

database. Then, the respective loop templates were grafted onto the designated light chain and heavy

chain frameworks, followed by energy minimization in the transition area between CDRs and frameworks

in order to relax strained geometries using AMBER12EHT force field. In order to gain confidence in the

modeled GSK2394000 and GSK2394002 Fab structures each was superimposed with its solved parental

murine crystal structure (7B4.1E11 and 12F4.1H7, respectively) as shown in Supplementary Figure 3.

Superimposed structures with a root mean square deviation (RMSD) less than 1.5Å from the murine

crystal structure were considered a good model. RMSD is the square root of the mean of the square of the

distances between the matched atoms in the two structures: RMSD = SQRT[{SUM(dii)2}/N], where dii is

the distance between the ithatom of structure 1 and the ith atom of structure 2 and N is the number of atoms

matched in each structure. The RMSD is 0 for identical structures, and its value increases as the two

structures become more distinct. RMSD values are considered as reliable indicators of variability when

applied to conformations of the same protein. RMSD comparisons for the superimposed structures: 7B4

(416 atoms) = 0.363Å and 12F4 (346 atoms) = 1.249Å. One exception was noted in the 12F4.1H7 crystal

structure where poor resolution for a portion of CDRH2 (spanning amino acids 53-68) led to an inability

to accurately compare this region. Without a matched parental crystal structure the 7E8.1E3 Fab structure

relied solely on computational modeling based on the variable region sequence (below) and comparison

to the available antibody structure database in PSILO.

Fab Antibody Amino Acid Sequence (CDRs)

GSK2394000 Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFTF

SDAWMDWVRQAPGKGLEWVAEIRNKANNHARHYAESVKGRFTISRDNAKNSLYLQMNSLRAE

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DTAVYYCARTYYYGSSYGYCDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSCDKTH

GSK2394000 Light chain

DIQMTQSPSSLSASVGDRVTIT

CRTSENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

QHHYGTPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

GSK2394002 Heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFTF

SDAWMDWVRQAPGKGLEWVAEIRHKANDHAIFYDESVKGRFTISRDDSKNTVYLQMNSLRAE

DTAVYYCTSPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK

TH

GSK2394002 Light chain

DIQMTQSPSSLSASVGDRVTIT

CKASQSVGTTIVWYQQKPGKAPKLLIYSASNRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

QQYTSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

7E8.1E3 Heavy Chain

QVQLQQSGAELVRPGASVTLSCKA

SGYTFTDYEIHWVKQTPVHGLEWIGPIDPETGNTAYNQKFKGKAIMTVDKSSSTAYMELRSLTSE

DSAVYYCTREGLRGHWYFDVWGAGTTVTVSSAKTTPPSDYPLVPGSAAQTNSMVTLGCLVKG

YFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKK

IVPR

7E8.1E3 Light Chain

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QIVLIQSPAIMSAFPGERVTM

TCSASLSVTYMYWYQQRPGSSPRLLISDTSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC

QQWSYYPVTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGS

ERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE

Structure best-fit modeling

High resolution structures of antibody Fab fragments and antibody-antigen complexes are useful for

analyzing the binding interface and to make rational choices for affinity maturation. Using a Rosetta

computational docking program [5] the structural complex is modeled to predict and characterize a

binding interface between two proteins. ADAMTS5-5 catalytic and disintegrin domain structure (PDB:

2RJQ), with small molecule inhibitor removed, was independently docked with the modeled humanized

versions of the 7B4.1E11 (GSK2394000) and 12F4.1H7 (GSK2394002) structures as described above.

ADAMTS-4 catalytic and disintegrin domain (PDB: 3B2Z) was similarly docked with the modeled

7E8.1E3 Fab. The first stage of the algorithm employs a rigid-body Monte Carlo search, translating and

rotating the antigen around the surface of the Fab, using residue-scale interaction potentials. An

alignment score directs the antigen toward the antibody CDR loops. After the low-resolution search,

explicit side chains are added to the protein backbones using a backbone-dependent rotamer packing

algorithm. A Monte Carlo-plus-minimization scheme then efficiently samples a set of local minima in a

small region of docking conformation space by simultaneously optimizing the side-chain conformations

and the rigid-body position resulting in identification of the top 200 best-fits. Ultimately, the top 10

binding predictions were overlaid and Fab orientation clustering was used to assess confidence in a single

preferential binding site and docking orientation. Models demonstrating a high degree of consistency for

the top 10 fits (within 2.5Å resolution) in the clustering around a specific region of the protease met the

confidence threshold and the top ranked binding prediction was progressed to bond energy analysis at the

protease/antibody interface, while those that did not (ADAMTS-4 with ADAMTS-5 mAbs, for example,

which demonstrated a wide distribution across the protease surface) were deemed inefficient binders,

consistent with the observed binding selectivity profile for these mAbs (manuscript Table 1 and Figure 1).

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Computational protease/antibody interface bond energy prediction

The top ranked docking prediction for the ADAMTS-5 mAbs (GSK2394000 and GSK2394002) with

ADAMTS-5 from above were used in an Alanine substitution scan for each predicted hydrogen bond site

to determine the importance of a residue to the overall stability and affinity of the interaction. Alanine

was used because of its side chain is non-bulky and chemically inert, but still has secondary structure

preferences similar to most amino acids. In the model, stabilizing or destabilizing energy relative to the

wild-type antibody was assessed computationally using the Rosetta program [5] and resulting change in

relative hydrogen bond energy (Kcal/mol) as shown (Figure 2c). If the predicted change in bond energy

is positive, the wild-type is contributing more favorably to the binding free-energy than the alanine

mutant, in contrast, if the energy change is negative, the mutant is contributing more favorably. The

magnitude indicates how important a particular energy estimator is to determining the relative free energy

change of the mutant compared to the wild-type.

Human OA Cartilage Explant Processing and Assessment of Treatment Efficacy

Cartilage was carved from bone, cut into uniform 3-mm diameter disks of similar thickness and cultured

[DMEM with 10% FBS, Pennicillin (10IU/mL), Streptomycin (10g/mL) and L-glutamine (0.29mg/mL)]

in 96-well plates. Tissue from a single donor was used in each plate. Following a 3-5 day culture

equilibration, media was removed and treatments were added without exogenous stimulation using a

continuous or transient (pulse-chase) treatment design. Each treatment was performed in replicates of

seven/plate. Supernatants were harvested twice weekly throughout the 3-week unstimulated phase when

half the well volume was removed (100L), stored frozen (-20°C) until analysis, and replaced with fresh

media containing either consistent treatment concentration (continuous design) or no treatment (pulse-

chase design). Immediately following, in most experiments, explants were stimulated with exogenous

human interleukin-1beta and oncostatin M [10ng/mL each]. Cartilage degradation in culture supernatants

was monitored using ARGS neoepitope detection. Percent inhibition was calculated compared to isotype

control and small molecule inhibitor (GSK571949) treated samples

Animal use compliance

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All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of

Laboratory Animals and were reviewed by Institutional Animal Care and Use Committees either at GSK

or Rush University.

Mechanical Allodynia

Male C57BL/6 mice (10 weeks of age) were pre-dosed with ADAMTS-5 (7B4.1E11) or isotype control

mAbs [10mg/kg] 3 days prior to DMM surgery followed by weekly maintenance doses and direct effects

on pain-related endpoints were assessed (supplementary methods). Throughout the 8-week study mice

were tested using the up–down staircase method [6]. Mice were placed on a perforated metal floor (with

5-mm diameter holes placed 7 mm apart) within small Plexiglas cubicles, and a set of eight calibrated von

Frey fibers (Stoelting Touch Test Sensory Evaluator Kit; ranging from ~0.04 g to ~2.0 g of force) were

applied to the plantar surface of the hind paw until the fibers bowed, and then held for 3 s. The threshold

force required to elicit withdrawal of the paw (median 50% withdrawal) was determined twice on each

hind paw (and averaged) on each testing day, with sequential measurements separated by at least 5 min.

Thresholds were assessed prior to surgery, and again 2, 4, and 8 weeks post-surgery.

Dorsal Root Ganglion (DRG) Immunofluorescence

Eight weeks post-DMM mice from the pain study described above were anesthetized by ketamine and

xylazine and perfused transcardially with PBS followed by 4% paraformaldehyde in PBS at the

completion of the study. The spinal column was dissected and postfixed in 4% paraformaldehyde

overnight followed by cryopreservation in 30% sucrose in PBS. Individual ipsilateral L3-L5 DRG were

embedded with OCT (Tissue-Tek), snap frozen, and cut into 12 μm sections. DRG sections were stained

with the primary antibody anti-F4/80 (Abcam ab6640) and isotype-specific secondary AlexaFluor-633

antibody (Invitrogen) [7]. All captured images were exported to Adobe Photoshop CS 5.1 (Adobe, San

Jose, CA) and adjustments were made to the brightness and contrast to reflect true colors.

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Supplementary Tables:

Supplementary Table 1: Demographics and circulating ARGS profile of cynomolgus monkeys tested

Age (Mean years, [95% CI Range]) 4.44 [4.007 to 4.874]Animals, n (% female) 31 (51.6)Weight All (Mean kg, [95% CI Range]) 4.023 [3.510 to 4.537]

Weight Male (n=15) 4.571 [3.620 to 5.522]Weight Female (n=16) 3.510 [3.102 to 3.918]

Serum ARGS (Mean ng/mL, [95% CI Range]) 7.122 [4.662 to 9.582]Serum ARGS Male (n=15) 11.19 [6.945 to 15.44]

Serum ARGS Female (n=16) 3.308 [2.379 to 4.236]

Supplementary Table 2: Statistical correlation analysis of various parameters in study monkeys

The relationships were analyzed using Pearson correlation method, correlation coefficient and p-value

were to be reported and positive value of correlation coefficient is indicative of positive correlation.

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Supplementary Figures:

--Intact Aggrecan IGD

--NITEGE373 IGD Fragment--mAb Heavy Chain and

374ARGS IGD Fragment

--mAb Light Chain

Sample Lane ID’s1 MW Ladder2 ADAMTS-5 (TS-5) Alone3 Aggrecan (IGD) Alone4 IGD + TS-55 TS-5 + GSK23940026 GSK2394002 Alone7-13 IGD + TS-5 + GSK2394002

kDa191

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19

1 2 3 4 5 6 7 8 9 10 11 12 13

1000

nM

500n

M

250n

M

125n

M

62.5

nM

31.3

nM

15.6

nM

GSK2394002 Concentration

--Intact Brevican

--Brevican Fragment and ADAMTS5

--mAb Heavy Chain and Brevican Fragment

-- mAb Light Chain

kDa191

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1000

nM

500n

M

250n

M

125n

M

62.5

nM

31.3

nM

15.6

nM

GSK2394002 Concentration1 2 3 4 5 6 7 8 9 10 11 12 13

Sample Lane ID’s1 MW Ladder2 ADAMTS-5 (TS-5) Alone3 Brevican Alone4 Brevican + TS-55 TS-5 + GSK23940026 GSK2394002 Alone7-13 Brevican + TS-5 + GSK2394002

Supplementary Figure 1: GSK2394002 inhibits ADAMTS-5-mediated proteolysis of multiple substrates.

Titration of GSK2394002 in the presence of ADAMTS-5 and either aggrecan IGD (top) or brevican

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(bottom) demonstrate dose-dependent inhibition of proteolysis (lanes 7-13). Individual assay controls in

each experiment (lanes 2-6) are shown for molecular weight comparison of individual components and

were treated similarly. Molecular weight markers are shown (lane 1). Identities of each fragment based

on respective control comparison are shown on the right.

Supplementary Figure 2: Pre-treatment ARGS neoepitope levels in individual human OA cartilage

explant cultures correlate with response to GSK2394002 treatment. A heat map depiction shows ARGS

neoepitope levels are elevated in approximately half of individual OA patient cartilage explants prior to

treatment. Background samples were taken immediately following treatment addition on day 0 while the

experimental phase (response to treatment) was assessed following a single 5 day treatment with 670nM

of GSK2394002 and isotype control mAbs. Values shown for pre-treatment, background and

experimental phase are average ARGS neoepitope levels from 7 biological replicates at each timepoint.

Note: although 17 independent experiments were performed in this study, two utilized separate samples

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from a single patient, 33745 and 33745a, to assess consistency of response in cartilage from an individual

patient.

Supplementary Figure 3: Superimposed alignments of modeled humanized and crystallized parental

murine ADAMTS-5 Fab structures support accurate modeling. (a) Overlay of 7B4.1E11 Fab crystal

structure (red) and computationally modeled humanized GSK2394000 Fab (green). (b) Overlay of

12F4.1H7 Fab crystal structure (red) and computationally modeled humanized GSK2394002 Fab (green).

Supplementary References

7B4.1E11 and GSK2394000 Superimposed

12F4.1H7 and GSK2394002 Superimposed

a. b.

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1. Deng H, O'Keefe H, Davie CP, Lind KE, Acharya RA, Franklin GJ, et al. Discovery of highly

potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage

degradation via encoded library technology (ELT). J Med Chem 2012; 55: 7061-7079.

2. Co MS, Queen C. Humanized antibodies for therapy. Nature 1991; 351: 501-502.

3. Pratta MA, Su JL, Leesnitzer MA, Struglics A, Larsson S, Lohmander LS, et al. Development

and characterization of a highly specific and sensitive sandwich ELISA for detection of

aggrecanase-generated aggrecan fragments. Osteoarthritis Cartilage 2006; 14: 702-713.

4. Germaschewski FG, Matheny, C., Larkin, J., Liu, F., Thomas, L., Saunders, J., Sully, K., Whitall,

C., Boyle, Y., Peters, G., Graham, N.M. Quantitation of ARGS aggrecan fragments in synovial

fluid, serum and urine from osteoarthritis patients. Osteoarthritis Cartilage 2014; 22: 690-700.

5. Lyskov S, Gray JJ. The RosettaDock server for local protein-protein docking. Nucleic Acids Res

2008; 36: W233-238.

6. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile

allodynia in the rat paw. J Neurosci Methods 1994; 53: 55-63.

7. Miller RE, Tran PB, Das R, Ghoreishi-Haack N, Ren D, Miller RJ, et al. CCR2 chemokine

receptor signaling mediates pain in experimental osteoarthritis. Proc Natl Acad Sci U S A; 109:

20602-20607.

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