| 1| 1| 1Club Phase I, 22 March 2016

Immunogenicity …How to deal with ?

Nathalie Macé

Sanofi, Biomarkers & Biological analyses Unit

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Outline

● Introduction to immunogenicity

● Analytical challenges for immunogenicity testing

● Non-clinical immunogenicity testing

● Clinical immunogenicity testing

● Approach

● Main parameters to be evaluated

● Risk assessment

● Application to a Phase I clinical trial

What is immunogenicity ?

● Immunogenicity is the propensity of biotherapeutics to generate a humoral

immune response (Anti-Drug Antibodies “ADAs”) to themselves and structurally

related molecules

● Terminology commonly used:

● ADA for Anti-Drug antibodies

● ATA for Anti-Therapeutic Antibodies (for immuno-conjugate)

● Two major mechanisms

● Reaction to neo-antigens (non human sequences, new epitopes, hypervariablessequences …)

● Breaking of immune tolerance (native aggregates)

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Immunogenicity and therapeutic proteins

● Why testing ?

● To detect and characterize a potential unwanted immune response during

development of therapeutic proteins in preclinical and clinical studies

● Immunogenicity can affect both efficacy and safety

● Impact on efficacy:

• Altered PK

• ADAs increasing the clearance of a biotherapeutic => reduced

efficacy

• ADAs decreasing the clearance of a biotherapeutic => problematic for

agonistic drugs => exaggerated pharmacology

• Impairment of target binding by neutralizing antibodies

● Impact on safety:

• Hypersensitivity reactions (prior sensitization)

• Anaphylaxis (type 1 hypersensitivity)

• Deposition of immune complexes in tissues (type 3 hypersensitivity)

• Neutralization of endogenous counterpart (immunodeficiency syndrome)

But in many cases ADAs do not have a clinical impact

when no endogenous counterpart

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Analytical challenges

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Pre-requisites in the implementation of an immunogenicity assay

● Be sufficiently sensitive to detect specific antibodies in study samples (250 –

500 ng/mL in human, 500 – 1000 ng/mL in animal – required range by the

Health Authorities)

• Quantitative assay (concentrations) qualitative assay (results are “status”)

● Have few false-positive (which will be then confirmed as false-positive or true

positive in confirmatory assay): 5% are required by the Health Authorities

● Detect ADA in presence of circulating therapeutic proteins = drug

interference Have no false-negative

● 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

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0.0

0.2

0.4

0.6

0.8

1.0

Response (

OD

450 n

m)

Pre

Week 2

Week 4

Week 6

Week 8

Week 1

2

Week 1

7

Week 2

5Wash-out

negative

Transient

positive

Drug InterferenceWash-Out Samples

Drug InterferenceAcid dissociation of the immune-complexes

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Detection without excess of drug Detection with excess of drug pH 2-3 acidification

of samples

Challenges in immunogenicity testing

● In contrast to PK, non availability of a « reference standard » necessity to obtain a « surrogate anti-drug antibody » qualitative assay

● Immunisation in animal species (rabbit, goat, sheep …)

• repeated administration of the therapeutic protein (hyperimmunisation)

● Purification step obtaining of a purified serum of polyclonal anti-drug

antibodies

● This surrogate control substantially differs from the measured human anti-drug

antibodies in respect to affinity and avidity

• consequently no exact numbers (e.g. for sensitivity) can be reported for

immunogenicity assays (but numbers relative to the batch of positive control used)Immunization of a goat with drug

Goat-serum

IgG goat-serum Goat-anti-drug

Goat-anti-human

antibodies (IgG)

Pro

tein

A c

olu

mn

dru

g c

olu

mn

Hu

ma

n I

gG

Iso

typ

eC

olu

mn

Goat anti-drug

antibodies (IgG)

If drug is an antibody

Immunization of a goat with drugImmunization of a goat with drug

Goat-serum

IgG goat-serum Goat-anti-drug

Goat-anti-human

antibodies (IgG)

Pro

tein

A c

olu

mn

dru

g c

olu

mn

Hu

ma

n I

gG

Iso

typ

eC

olu

mn

Goat anti-drug

antibodies (IgG)

If drug is an antibody

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Assay formats

Particularity: Bridging formats

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ELISA bridging

Electrochemiluminescence bridging

Quality of reagents is very important

Validity of results monitored by a system of positive controls

● Establish a suitable system of control to ensure the validity of results

● A performance control set (PC) must be defined and should include:

• Negative control (NC): pool of naïve individual healthy subjects

• Positive controls: pool of individual naive matrices spiked with «surrogate ADA»

● Define acceptance criteria for PCs

● PC set will be used to validate each run

Screening cut-point

: positif PCs

: negatif PC

HiPC

LLPC > SCP (<250-500 ng/mL, human)

Negative PC < SCP

PCs

LoPC

MiPC

Sig

na

l

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Non-clinical immunogenicity

testing

Animal Studies

Interest of immunogenicity testing

● The immune response of different animal species to a human therapeutic

protein is not considered being predictive for immunogenicity in

humans

● Primary aim of non-clinical immunogenicity testing is to aid in the

interpretation of toxicity results (relationship between dose and

exposure) but not to ensure safety of the tox species

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Clinical immunogenicity testing

Strategy for the evaluation of immungenicity is based on regulatory documentation:

- FDA Guidance for Industry (2009): Assay development for Immunogenicity Testing of Therapeutic Proteins. Draft.

- FDA Guidance for Industry (2014): Immunogenicity Assessment for Therapeutic Proteins Products.

- EMA (2015) Guideline on Immunogenicity Assessment of Biotechnology-derived Therapeutic Proteins. EMEA/CHMP/BMWP/14327/2006 Rev. 1

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Clinical immunogenicity testing strategy3-tiered approach

Negative

Screening assay

Reactive

Neutralizing Assay

Characterization

Confirmatory assay Negative

Confirmed Positive Titer

Tier 1: 5% false-positive

ADAs present?

Tier 2: Are the detected

ADAs specific for the drug?

Negative

Confirmed Positive Titer

Tier 3: Do the specific

ADAs possess neutralizing

capacity?

Correlation ? with

clinical observations

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How to determine the screening cut point (SCP)?

● SCP = level of response above which a sample is defined to be reactive(potential positive) and below which is probably negative

● Upper negative limit of 95% is recommended = risk-based approach (more appropriate to have 5% false-positive than false-negative)

Sample No.

0 5 10 15 20 25 30 35 40 45 50

OD

45

0 n

m

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Cut Point = NSB + 1.645 x SD

Mean Response (=NSB)

Sample No.

0 5 10 15 20 25 30 35 40 45 50

OD

45

0 n

m

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Cut Point = NSB + 1.645 x SD

Mean Response (=NSB)

● Normalization of screening cut point

● fixe SCP Floating cut point determination of a correction factor = CF

● CF allows taking into account the inter-run variability

● Statistical approach

How to determine the specificty cut point ?

● Used to confirm the presence of ADA in sample found above the SCP● Competitive inhibition by addition of the therapeutic protein in sample

● % inhibition calculated between sample measured unspiked and spiked with an excess of drug

● Sample is confirmed positive if his %inhibition is > %inhibition

● 0.1% of false-positive

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Pat 1 Pat 2 Pat 3 Pat 4

Response

0.0

0.2

0.8

1.0

Unspiked

Spiked

Pat 1 Pat 2 Pat 3 Pat 4

Response

0.0

0.2

0.8

1.0

Unspiked

Spiked

Pat 1 Pat 2 Pat 3 Pat 4

Response

0.0

0.2

0.8

1.0

Unspiked

Spiked

Confirmed

Positive

Confirmed

Positive

Negative

Important: Suitability of the Cut-

point needs to be re-assessed

once baseline samples from

patients become available

Measured signal > SCP

=> Reactive sample

Concentrations croissantes du BT

Surface immobilisée avec le BT

Concentrations croissantes du BT

Surface immobilisée avec le BT

Addition (spike) of an excess of drug in sample

Concentrations croissantes du BT

Surface immobilisée avec le BT

Concentrations croissantes du BT

Surface immobilisée avec le BTDecrease of measured signal

=> Sample confirmed as positive if %inhibition > CCP

● Definition● Lowest concentration at which a positive control antibody preparation

consistently provides a positive signal for the presence of ADAs

● Sensitivity of the ADA assay is assessed using serial dilutions of the positive

control spanning the assay cut-point

● Health Authorities expect an ADA assay for the clinical setting to at least

achieve a sensitivity of 250-500 ng/mL

● Of note: Basic serum IG concentration 15 mg/mL

SCPplate

ADA concentration

Signal

Serial dilution of

positive PC

i.e. 10 µg/mLi.e. Sensitivity=250 ng/mL

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Sensitivity

● The presence of major amounts of drug interferes with the

detection of anti-drug antibodies leading to “false negatives”

● To determine the drug tolerance of an ADA assay:

• Low or Lower (at the sensitivity level) positive control is pre-incubated

with increasing amounts of drug

• The highest drug concentration that still results in a response above the

assay cut-point will be defined as drug tolerance level

Concentration Drug (µg/mL)

0 2 4 6 8 10 12 14 16 18

OD

45

0 n

m

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Cut-Point

HPC

LPC

Drug Tolereance HPC

Drug Tolereance LPC

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Drug tolerance

Always associate a PK time point for

the interpretation of immunogenicity

results

(essentially in early phases to

determine the immunogenicity profile)

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Titration

● Aim:

● Retrieve quasi-quantitative information for confirmed positive samples

● Procedure:

● Serial dilution of confirmed positive samples

● Titer = defined as the reciprocal of the highest dilution of the sample

(including MRD) that yields a positive signal result

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 1/100=> Titer = 100

Neutralizing Antibodies - Why Testing for…? Regulatory perspective

Neutralizing antibodies (NAbs) block the biological activity of the therapeutic

Direct binding to the epitope within the active site

Block active site by steric hindrance

● Neutralizing antibodies can affect both efficacy and safety

● Impact on efficacy:

• Impairment of target binding by neutralizing antibodies

● Impact on safety:

• Neutralization of endogenous counterpart (immunodeficiency syndrome)

● Assessment of the neutralizing capacity of detected ADAs has to be considered as

part of all clinical trial protocols

● Analytical challenge:

● Assay for the detection is very sensitive detection of very low

concentrations of ADA difficulties to show their neutralizing capacities

and clinical impact

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Immunogenicity risk

assessment

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Immunogenicity Risk Assessment

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Risk = Severity x Probability

Consequences of ADAs Incidence of ADAs

Impact on safety Impact on efficacy

● Prediction of immunogenicity is distinct from risk assessment

● Prediction tools might only help predicting the probability of an ADA

response but not its clinical sequelae

Immunogenicity Risk AssessmentClassification into Immunogenicity Risk Categories

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Lower risk Medium risk Higher risk

Clinical sequalae of ADAs HypersensitivityNone Exaggerated

pharmacology due to

ADAs decreasing the

clearance

Immunodeficiency

Factors

Primary sequenceFully human Non human Any Any

Glycosylation

patternAny Any Any

Unique endogenouscounterpart

- - - /-

Mode of actionAntagonistic Any Agonistic Any Any

Alternative treatmentavailable

/- -

Impurities - /- /- /-

Degradationproducts/ Posttranslationalmodifications

- /- /- /-

High aggregatecontent - /- /- /-

Loss of efficacy in

life threating disease

with no alternative

treatment

Fully human Non human

Fully human

Green: All critera need to be fulfiled for classification

Red: One criterion is sufficient for classification

Risk Based Immunogenicity Testing Strategy

Testing strategy for

lower / medium risk proteins

Testing strategy for higher risk

proteins

Frequency of sampling within study:

• More frequently early in the drug program

and less frequently in phase III trials

Assessment of ADAs:

• Detection of ADAs using screening and

confirmatory assays

• Titration of confirmed positive samples

• Assessment of the neutralizing capacity of

ADA positives may be explored as

necessary

• Consider drug specific IgE testing in case of

hypersensitivity reactions

Sample testing:

• Retrospective analysis at the end of a trial

might be sufficient

Frequency of sampling within study:

• More frequently throughout all phases of

clinical development

Assessment of ADAs:

• Detection of ADAs using screening and

confirmatory assays

• Titration of confirmed positive samples

• Assessment of the neutralizing capacity of

ADA positives

Sample testing:

• Consider real time analysis of ADA

samples and post-study follow-up of

positive subjects

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Application to a Phase I clinical

study

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Presentation of the project - History

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● A FIM study in patients began in 2010

• Anticipated concentrations of circulating drug were very low

• Wide range of doses tested => very high concentrations of circulating drug in patients

● Chronology of this project in term of immunogenicity

• Based on EMA guidance and FDA draft guidance first validation performed with a

bridge ELISA format

• Determination of the SCP and CCP:

• First on naive healthy subjects,

• then when available on patient matrices

• Drug tolerance (DT) of the assay: 100 µg/mL

• Compliant with expected PK Cmax and doses scheduled at the beginning of the

study

Analysis of study samples started with the validated method

Consequences of ADA wording harmonization …

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● Release and implementation of ABIRISK and AAPS papers for harmonization

of reporting of ADA results

● Final status of sample is linked to the concentration of circulating drug

• Sample with negative status and concentration of circulating drug > DT of

the assay sample has to be reported as « inconclusive »

• Status of all analysed samples were re-evaluated compared to the

concentration of drug in sample => ~ 30% of samples became

« inconclusive » only the positive results are really interpretable

● Drug is well tolerated by patient increase of doses

● Faced to concentrations of circulating drug > DT

• Not possible to calculate the incidence of immunogenicity

• If clinical sequelae => not possible to correlate with a status

• Not more possible to evaluate the impact

Not acceptable by the Authorities

Action plan

● Verification if the actual method could be improved in term of DT

● Not possible to improve the DT with the ELISA bridge

● Development and validation of an innovative method

● Improvement of drug tolerance (cover the circulating concentration of drug)

● Keeping at least the same sensitivity

● Analysis of study samples in parallel with the 2 methods (bridge ELISA and

new method)

● Excellent concordence of results between the 2 methods

● Submission of this new method to the Health Authorities with clinical results

obtained with the 2 methods

• Proposal to stop analyses with the ELISA bridge method and to switch on the

new method acceptation from Authorities to consider the new method as

the reference method

• Consequences: no more samples have an inconclusive status and

possibility to calculate the incidence and correlate with potential clinical

impact

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THANKS!!!!!!

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