Binding Antibodies: Assay Methodologies, Screening ... · Binding Antibodies: Assay Methodologies, Screening Confirmation, Characterization of Anti-Drug-Antibodies EIP Open Symposium

Binding Antibodies: Assay Methodologies, ScreeningConfirmation, Characterization of Anti-Drug-Antibodies

EIP Open Symposium Kopenhagen 2012

Daniel Kramer, Global DMPK, Merck Serono, Germany

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Immunogenicity Testing – How…?

Putative PositivePutative Positive NegativeNegative

Tier 2: Confirmatory AssayImmune competition to eliminate false positives

Tier 2: Confirmatory AssayImmune competition to eliminate false positives

Confirmed PositiveConfirmed Positive

Tier 3: Characterization AssaysTitration; Neutralizing capacitiy of binding antibodies

Tier 3: Characterization AssaysTitration; Neutralizing capacitiy of binding antibodies

NegativeNegative

Tier 1: Screening AssayIdentification of putative positive samples based on cut-point

Tier 1: Screening AssayIdentification of putative positive samples based on cut-point

3

1st Tier: Screening AssayRadio-Immunoprecipitation (RIP)

Add iodine-labeled drug to serumcontaining anti-drug antibodies

Add protein Acoated beads

Centrifugemeasure pellet

4

1st Tier: Screening AssayRadio-Immunoprecipitation (RIP)

Advantages– Sensitivity

– Rather high drug tolerance

Disadvantages:– Low throughput

– Restricted availability of CROs

– Specificity (prone to artefacts)

– Radiolabelling process can mask/denature epitopes recognized by anti-drug antibodies

– Protein A/G are known of having different affinities to different isotypes

5

1st Tier: Screening AssayDirect ELISA

Immobilize drug Add serum containinganti-drug antibodies

Detect with enzyme labeledpolyclonal secondary

antibody

6

1st Tier: Screening AssayDirect ELISA

Advantages:– Sensitivity

– Commercial available secondary antibodies

Disadvantages:– Source of the positive control has to be the same as that of the anti-

drug antibodies

– Specificity (unspecific binding to matrix components)

– Restricted detection of low-affinity antibodies

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1st Tier: Screening AssayBridging ELISA

Immobilize drug Add serum containinganti-drug antibodies

Detect with labeled(e.g. biotinylated)

drug

8


Advantages– High throughput

– Specificity (two-fold binding of drug required for signal)

– Possibility to use any positive control binding to the drug (independent of species)

Disadvantages– Sensitivity (special orientation of immobilized drug required)

– Restricted detection of low-affinity antibodies

– Biotinylation might mask/denature epitopes recognized by anti-drug antibodies

9

direct ELISA bridging ELISA

OD

450

nm

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

before treatment after treatment


Sensitivity

10

Time [s]

0 100 200 300 400 500 600 700

Res

pons

e [R

U]

800

1000

1200

1400

1600

1800

2000

2200

Drug Biotin labeled Original drug

Masking of binding epitopes by biotin


11

1st Tier: Screening AssayElectrochemiluminescence (ECL)

Ruthenium (II) Sulfo-tris-bipyridine NHS ester= sulfoTAG

Incubate sample withbiotin-labeled drug

andsulfoTAG labeled drug

Transfer solution to pre-blocked MSD streptavidin plate

Wash assay plate;add Read Buffer; Read plate

electrode

sulfoTag emits light whenelectrochemically stimulated

12


Advantages– Electrochemiluminescence technology offers sensitivity and large

dynamic range

– Less washing steps allow the detection of low affinity anti-drug antibodies

– Better tolerance for drug than ELISA

– Possibility for multiplexing (epitope mapping)

Disadvantages– The use of two conjugated reagents increases the risk of masking of

binding epitopes

13


Sulfo-TAG-Drug

Biotin-Drug

Detectable in MesoScale

Sample containingexcess of drug

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Drug InterferencePositive samples were spiked with increasing amounts of drug and analyzed in Mesoscale and (bridging) ELISA

Spiked drug concentration [µg/mL]

0 1 2 3 4 5 6

Sig

nal [

%]

0

20

40

60

80

100

ELISA Mesoscale

Low anti-drug antibody concentration

Spiked drug concentration [µg/mL]

0 1 2 3 4 5 6

Sig

nal [

%]

0

20

40

60

80

100

ELISA Mesoscale

High anti-drug antibody concentration

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Drug immobilized on sensorchip

Injection of serum containing anti-drug antibodies

800900

10001100120013001400150016001700

0 60 120 180 240 300 360 420 480 540 600 660

Response(RU) Sensorgram

Time (seconds)

Prisma

Gold-coated sensorchip

Flow channel

1st Tier: Screening Assay Biacore

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Immobilizedrug

Inject serumcontaininganti-drug

antibodies

Inject drug

Glass support

Gold layer

Inject anti Ig

antibodies

1st Tier: Screening AssayBiacore

17

800900

10001100120013001400150016001700

0 60 120 180 240 300 360 420 480 540 600 660

Response(RU)

Time (seconds)

On-rateka

Off-ratekd

Amount bound

Bulk effect

Bulk effect


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Advantages:– Large dynamic range

– No secondary reagents required

– Detection of low affinity antibodies

– Sensograms include information about affinity of anti-drug antibodies

– Easy procedure for isotyping

– Easy procedure for epitope mapping

Disadvantages:– Structure of drug might be influenced by chemical coupling

– Less sensitive than ELISA

– Time consuming

– Costs

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Sensograms contain information about affinity of anti-drug antibodies

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

0 100 200 300 400 500 600 700 800 900

Time s

RU

-500

0

500

1000

1500

2000

2500

0 100 200 300 400 500 600 700 800 900

Time s

RU

Fast off-rate

Slow off-rate


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Sensitivity / Dynamic range

c [µg/mL]

0.001 0.01 0.1 1 10 100

RU

0

200

400

600

800

1000

1200

1400

1600

1800

2000

OD

450 nm

0.0

0.5

1.0

1.5

2.0

BiacoreBridging ELISA

250 ng/mL


21

Spiked drug concentration [ng/mL]

0 1000 2000 3000 4000 5000 6000

Sig

nal [

%]

0

20

40

60

80

100

Bridging ELISABiacore

Low anti-drug antibody concentration

Spiked drug concentration [ng/mL]

0 5000 10000 15000 20000 25000

Sig

nal [

%]

0

20

40

60

80

100

Bridging ELISA Biacore

High anti-drug antibody concentration

Positive samples were spiked with increasing amounts of drug and analyzed in Biacore and (bridging) ELISA

Drug Interference


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Sampling dayPre day 5 day 8 day 16 day 22

Rel

ativ

e co

ncen

tratio

n [n

g/m

L]

0

200

400

600

800

1000

1200

1400

1600

Biacore Bridging ELISA

Low affinity antibodiesAdvantage: Biacore

High affinity antibodiesAdvantage: ELISA(higher sensitivity)

Low Affinity Antibodies


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Epitope Mapping

Flow Cell 1

Flow Cell 2

A immobilized

B immobilized

Sample

Fusion proteinA-B

Drug:

Time [s]0 200 400 600

RU

-150000

-100000

-50000

0

50000

100000

150000

FC 1

Time [s]

0 200 400 600

RU

-150000

-100000

-50000

0

50000

100000

150000

FC 2

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1st Tier: Screening AssayGyros

25

ALEXA-Drug

Biotin-Drug

Spin

Serum containinganti-drug antibodies

Fluorescentsignal


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Advantages:– The Gyros technology offers sensitivity and large dynamic range– Detection of low affinity antibodies (homogenous format)– Rather high drug tolerance (homogenous format)– Requires only small sample volumes– Epitope mapping possible– High throughput– Automatization reduces variability (less manual pipetting steps)

Disadvantages:– The use of two conjugated reagents increases the risk of masking of binding

epitopes– Costs– Carry over


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1st Tier: Screening AssayCut-Point

Determination of the „non specific background“ (NSB) by testing of 50 serum samples of untreated animals or patients on three different days

Cut-Point: NSB + 1.645 x Standard Deviation (=> 5 % false positives)

Positive: Response > Cut-Point

Sample No.

0 5 10 15 20 25 30 35 40 45 50

OD

450

nm

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Cut Point = NSB + 1.645 x SDMean Response (=NSB)

Sample No.

0 5 10 15 20 25 30 35 40 45 50

OD

450

nm

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Cut Point = NSB + 1.645 x SDMean Response (=NSB)

Problem: Immunogenicity is a relative thing => criteria for positive samples needed

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1st Tier: Screening AssaySome Statistics

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Problem: Usually assay signal will vary between runs => Cut-Point normalization necessary

Normalization factor = relative response Cut-Point – relative response negative control

For each batch the normalization factor is added tothe relative response of the negative control to set the cutpoint

1st Tier: Screening AssayNormalization of Cut-Point

Sample No.

0 5 10 15 20 25 30 35 40 45 50

OD

450

nm

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Cut Point

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2nd Tier: Confirmatory AssayDue to the 5% false-positive rate built into the screening cut point, samples showing a response at or above the assay cut-point can just be considered “putative positive” for the presence of BAbs.

The confirmation of true positives among the putative positive samples requires the demonstration of specific binding to the drug: – A putative positive sample is re-tested in the presence and absence of an excess of drug.

– The specificity cut point is defined as the percent inhibition at or above which a sample is considered as “confirmed positive”.

Pat 1 Pat 2 Pat 3 Pat 4

Res

pons

e

0.0

0.20.8

1.0

UnspikedSpiked


Res

pons

e

0.0

0.20.8

1.0

UnspikedSpiked


Res

pons

e

0.0

0.20.8

1.0

UnspikedSpiked

ConfirmedPositive

ConfirmedPositive

Negative

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2nd Tier: Confirmatory AssaySpecificity Cut-Point

Spike all individual samples from the cut-point determination (preferable in the same experiment) with an excess amount of drug and calculate the percent inhibition per sample: 100 x [1-(spiked/unspiked)]

Calculate the specificity cut-point from the percent inhibition of all samples:Upper bound of a one-sided 99.9 % prediction interval (parametric: mean + 3.09 x SD or non-parametric: 99.9th percentile)

A real sample in study showing a higher % inhibition after spiking of drug than the specificity cut-point is defined as „confirmed positive“

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3rd Tier: TitrationAim:– Retrieve quasi-quantitative information for confirmed positive

samples

Procedure:– Serial dilution of confirmed positive samples

– Titer = -log dilution factor of the last dilution that tests positive

Dilution

1e-5(1:100000)

1e-4(1:10000)

1e-3(1:1000)

1e-2(1:100)

1e-1(1:10)

OD

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Cut Point

Dilution 1:2560 (= 3.91 x 10-4)=> Titer = 3.41

Dilution

1e-5(1:100000)

1e-4(1:10000)

1e-3(1:1000)

1e-2(1:100)

1e-1(1:10)

OD

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Cut Point

Dilution 1:2560 (= 3.91 x 10-4)=> Titer = 3.41

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Challenges In Immunogenicity TestingPositive Control

In contrast to PK assays the analyte is not available in purified formSerum from animals (e.g. goats) hyperimmunized with the drug is used as control insteadThis surrogate control substantially differs from the measured human anti-drug antibodies in respect to affinity and avidityConsequently no exact numbers (e.g. for sensitivity) can be reported for Immunogenicity assays (but numbers relative to the positive control)

Immunization of a goat with drug

Goat-serum

IgG goat-serum Goat-anti-drugGoat-anti-humanantibodies (IgG)

Prot

ein

A co

lum

n

drug

col

umn

Hum

an Ig

GIs

otyp

eC

olum

nGoat anti-drugantibodies (IgG)

If drug is an antibody

Immunization of a goat with drugImmunization of a goat with drug

Goat-serum

IgG goat-serum Goat-anti-drugGoat-anti-humanantibodies (IgG)

Prot

ein

A co

lum

n

drug

col

umn

Hum

an Ig

GIs

otyp

eC

olum

nGoat anti-drugantibodies (IgG)

If drug is an antibody

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Challenges in Immunogenicity TestingDrug Interference

The presence of major amounts of drug interferes with the detection of anti-drug antibodies and leads to “false negatives”

Solutions:– Wash-Out Samples

• Draw blood samples for the detection of anti-drug antibodies several days/weeks after the last treatment (5-6 x t1/2)

• Acid dissociation of the immunecomplexes

Serum samplewith excess

of drug

ELISA plate withimmobilized drug

No signal

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Wash-Out Samples

Draw blood samples for the detection of anti-drug antibodies several days/weeks after the last treatment (5-6 x t1/2)

Problem: A transient immune response might not be detected in wash-out samples => acid dissociation assays might be needed

0.0

0.2

0.4

0.6

0.8

1.0

Res

pons

e (O

D 4

50 n

m)

Pre

Week 2

Week 4

Week 6

Week 8

Week 12

Week 17

Week 25

Wash-outnegative

Transientpositive

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ACE Acid Dissociation Assay

Serum samplewith excess

of drug

Acidify

ELISA plate withimmobilized drug

and buffer

Detect withlabeled drug

Acidify

TransferNeuralize

Wash Transfer SupernatantNeuralize

Wash

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THANK YOU !

Documents

Binding Antibodies: Assay Methodologies, Screening ... · Binding Antibodies: Assay Methodologies, Screening Confirmation, Characterization of Anti-Drug-Antibodies EIP Open Symposium