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When reagents and technology matter to measurelow abundant protein biomarkersHervé Farine -Translational Biomarkers -19.09.2019

2IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcomeValidationDefine the purposeAssay selectionAssay developmentFeasibility studyIL-5NFLCXCL11

3IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcomeCOUIdentify the type 2 asthma patients (stratification)Biostatistician & translational scienceMeasurement of IL-5 in human serum samples of asthmatics (commercial assays available)

The assay selection based on LLOQ 4IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome0.5 pg/ml2014Mesoscale x-plex0.2 pg/ml2015Mesoscale V-plex2015Singulex/SMC2015ELLA1 pg/ml0.069 pg/ml

Only 2 assays allowed to measure the entire sample set.5IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome23%53%100%100%0%20%40%60%80%100%120%Mesocale X-plexMesocale V-plexSingulexELLA% of sample measured above LLOQ

Bad correlation with Singulex and strong correlation between Mesoscale V-plex and ELLA6IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome

0.1110

0.1 1 10

IL-5 [pg/ml]

Mesoscale V-plex

I L 5 p g m l E L L A

Spearman r: 0.77

0.1110

1 10 100

IL-5 [pg/ml]

ELLA I L 5 p g m l S i n g u l e x

Spearman r: -0.03

IL-5 ELLA was in the type 2 biomarkers cluster but not IL-5 Singulex7IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcomeIL-5 Sing.FVCACTFEV1PEFMIP-1bFeNOEDNIL-13Blood EosIL-5 ELLAIL-8TARCIL-6TNF-aIFN-gIP-10IL-5 ELLAIL-5 SingulexType 2 / eosinophilic

4 years later the COU changed !!8IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome•PD Biomarker•Sample: serum and nasal fluid•The assays changed:-ELLA: LLOQ

69 fg/ml à130 fg/ml

-Merck(Singulex) launched a new high sensitivity (HS) assay (with new antibodies)

LLOQ: 40 fg/ml

Singulex/SMC HS has a strong correlation with ELLA in serumand was able to measure 80% of the nasal fluid samples.9IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome

0.1110

0.1 1 10

IL-5 [pg/ml]

Singulex HS

I L 5 p g m l E L L A

Spearman r: 0.95

23%80%0%10%20%30%40%50%60%70%80%90%100%ELLASingulex HS% of nasal fluid sample measured above LLOQ

10IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcomeCOUPD effect for rodent model and human disease effect Translational science and preclinical pharmacologyDevelop an assay on MSD able to measure NFL in human and rodent in plasma and CSF

11IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome

19 healthy

donors 1 10 100
1000
N F L p g m l

0510152025

0 200
400
600
800
1000
1200
A d j R e s u l t N F L p g m l neat with heteroblock

Dilution factor

The addition of heterophilic antibodies blocker during the sample incubation reduced the level of the highest samples.12IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome

19 healthy

donors 1 10 100
1000
N F L p g m l

0510152025

0 200
400
600
800
1000
1200
A d j R e s u l t N F L p g m l neat with heteroblock

Dilution factor

The good correlation between human CSF and plasmashowed the specificity of the assay.13IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome

010000200003000040000

0 100
200
300
CSF

NFL [pg/ml]

P l a s m a N F L p g m l

Spearman r: 0.75

The use in mouse EAE model showed PD effect and a strong correlation with the pathology score.14IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome

naïv evehicletreatment 1 treatment 2 treatment 1 + 2 0 10000
20000
30000
40000
N F L p g m l

010000200003000040000

0 1 2 3 4 5

NFL [pg/ml]

P a t h o s c o r i n g

15IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcomeCOUPD effect in mouse modelPreclinical pharmacologyDevelop an assay able to measure CXCL11 in mouse plasma with 2 different high sensitivity platforms (Erennaand SR-X) with the same antibodies and the same standard.

Similar assay setup but 2 different technologies 16IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcomeErennaCXCL11MagneticBeadsCapture mAbDetectionpAbFluorescencesignalLLOQ = 1 pg/mlLLOQ = 0.4 pg/mlSR-X

SR-X showed the same trend than Erennabut with lower absolute values17IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome

C X C L 1 1 p g m L

VehicleTreatmentVehicleTreatment

0 20 40
60
80
SR-X

Erenna

The PD effect has the same amplitude in both assays.18IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcome

C X C L 1 1 F C t o v e h i c l e 0 1 2 3 4

SR-XErenna

19IntroductionIL-5 measurementNFL measurementCXCL11 measurementOutcomeTechnology improvesthe sensitivityProve the specificity of the antibodiesMinimize matrix effectMultiple comparison (biomarkers and assays)Be aware that immunoassays do not provide absolute values

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