· web viewfinal] was performed in individual tubes for 30 minutes to allow for binding. following...
TRANSCRIPT
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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