“Biotherapeutic Medicines – regulatory challenges and current practices. Approaches for harmonization” Moscow, Russia May 16, 2013. IFPMA/AIPM

Immunogenicity testing of Biotechnology Products and the Impact to Biosimilars

Barry Cherney

Executive Director Product Quality

Amgen Inc

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• Administration of non native or even ‘humanized’ proteins to either animals or humans can elicit either an antibody response, cellular response, or both if the immune system recognizes the protein as foreign.

• All protein products have some level of immunogenicity, with varying impact to patients.

• Although there are multiple theoretical causes of immunogenicity, very few real examples of why a product is immunogenic have been published

Protein Products have Immunogenic Potential

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Generalized Protein Domains

Receptor binding/functional domain

Non-receptor binding/functional domain

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Effect of antibodies on the function of proteins

Cell Response Neutralizing antibodies

Non-neutralizing antibodies

No cell response

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Clinical Concerns in Testing for Antibodies - Effects on PK/PD

• The binding of antibodies to product has been shown to potentially affect (by extension or reduction) the half life in blood through the influence on clearance mechanisms.

• Biodistribution of product has also been shown to be affected, such as lack of targeting to skin or tumour sites.

• If product is cleared differently and its distribution is different, then its ability to have the desired biological effect may also be altered.

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Clinical Concerns in Testing for Antibodies - Effects on Efficacy

• Antibodies that alter the PK/PD of the product may have an effect on its efficacy. If the drug remains longer in the circulation, efficacy can be enhanced. If half life is reduced, so potentially, is its efficacy.

• The presence of neutralizing antibodies can directly inhibit the biological activity of the product and thus clinical efficacy may be reduced or abrogated.

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Clinical Concerns in Testing for Antibodies - Effects on Safety

• Extending the half life of a product can influence its toxic properties.

• Redistributing a product to different sites may potentially also have safety implications.

• The presence of complexes of product and antibody can have physiological consequences.

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• Immune responses to product can lead to:• Anaphylaxis• Injection site reactions• Flu like syndromes• Allergic responses

• One of the most serious adverse events occurs when neutralizing antibodies to product cross react with endogenous proteins that have a unique physiological role.

Clinical Concerns in Testing for Antibodies - Effects on Safety

• “Comparative assessment of unwanted immune responses against the biosimilar and the reference mAb are normally undertaken as part of the clinical study”*

• “A risk-based approach can provide a starting point from which the further concept of immunogenicity testing can be designed, but due to the diversity of risk factors, as discussed in this guideline, and the variety of mAbs and mAb-related products, the recommendations given here cannot be generalized.”**

• “Assessment is based on the identification of risk factors inherent to the particular mAb in question, the final drug product and the treated patient population. The mechanism of action and the basic structure (chimaeric, humanized, fully human) are not sufficient for deciding on the attribution of risk level. For a risk-based approach, applicants need to define what “risk” in this context means.”**

* Guideline on similar biological medicinal products containing monoclonal antibodies – non clinical and clinical issues CHMP 2010 ** Guideline on immunogenicity assessment of monoclonal antibodies intended for in vivo clinical use. CHMP Nov. 2010

EMA mAb biosimilar and immunogenicity guidelines on the “risk-based approach”

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• “At least one clinical study that includes a comparison of the immunogenicity of the proposed product to that of the reference product will generally be expected “

• “The extent and timing …..of the clinical immunogenicity program will vary depending on a range of factors including the extent of analytical similarity and the incidence and clinical consequences of immune response for the reference product. “

*Scientific Considerations in Demonstrating Biosimilarity to a Reference Product FDA. Feb 2012

FDA’s draft biosimilar guideline* also includes “a risk based immunogenicity approach”

Risk Based Approaches to Immunogenicity Testing

Risk = Probabilityharm x Severityharm

• Severity outweighs the probability of a risk occurring.

• The overall Risk Score depends on an assessment of the various factors that influence immunogenicity

How many patients are likely to mount an immune response?

What happens to the patient if they mount an immune response?

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Considerations in Assessing Risk of Immunogenicity - Probability Analysis

Likely Lower Probability Likely Greater Probability

Immunosupressed patients Autoimmune disease

Single dosing Chronic dosing

More ‘Human’ ‘Foreign’

IV administration Subcutaneous

Highly pure Impure

No aggregates Aggregates

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Risk = Probabilityharm x Severityharm

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Considerations in Assessing Risk of Immunogenicity - Severity Analysis

Likely More Severe Likely Less Severe

Endogenous version No endogenous version

Unique activity Redundant activity

Sole therapy Other therapies

Life threatening disease Non life threatening disease

Chronic disease End stage disease

Non reversible AE Reversible AE

Replacement therapy Non replacement therapy

Anaphylactic response Non anaphylactic response

Risk = Probabilityharm x Severityharm

Examples of Anti Drug Antibodies: Incidence and Clinical Impact

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Rate of Antibodies

MoleculeAntibody Incidence

Clinical Impact

High r-Human alpha-galactosidase

88% none reported

High r-chimeric anti-TNFa

10-57%Hypersensitivity

PK affected efficacy unchanged

Moderater-chimeric

anti-GPIIb/IIIaFab

7-19%Higher incidence after

re-administration,TCP higher in ab+

patients

Moderate r-HumanGlucocere-brosidase

13%Neutralizing antibodies

rare

Lowr-Human

Thrombopoietintruncated, PEG

0.5-4%neutralizing antibodies

TCP

Low

r-Human TissuePlasminogen

Activator<1% none reported

Considerations for Analytical Testing Strategies based on the outcome of the Risk Assessment Score

Examples of High score considerations

Examples of Low score considerations

Test samples in real time Batch test samples at the end of the study

More sensitive assays early in development

Develop increasingly sensitive assays during development

Highly conservative approach to setting cut points

Less conservative approach to setting cut points

Test positive binding samples for neutralization in real time

Batch positive binding samples after initial screening and then

test for neutralization

Need to develop sensitive neutralization assays

Less need for very sensitive neutralization assays

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Biosimilar Specific Issues with Immunogenicity Testing

• The rate of immunogenicity detected in an assay is wholly dependent on the assay chosen, its controls, execution and the clinical study sample plan design

• Biosimilar companies are unlikely to know the details of the innovator assays:

• Assay format (RIA, ELISA - bridging or direct etc.)• Assay sensitivity and specificity• Assay positive control (standard)• Positivity criteria• Timing and number of samples• Patient population

• Multiple indications(if going into licensure with only one)

Google.www

The Optimal Screening Assay

An Optimal screening assay should:• Be sensitive enough to detect any level of

specific antibody present in patient sera.• Have no false negatives• Have a few false positives able to be

proved false positives• Discriminate between pre-existing

antibodies and treatment induced antibodies

• Detect antibodies in the presence of drug• Be reproducible

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The Choice of Assay Format can Heavily Influence the Rate of Immunogenicity Results Reported

Enzyme

Colouredreagent

Clearreagent

Detectingantibody

Anti-drug antibody

Drug

Serum Antibody

ConjugatedDrug

Drug

DetectionSystem

Add Serum Sample

Precipitate antibodybound drug

Measure radioactivityin pellet

ELISA

BridgingELISA

RadioImmunoprecipitationAssay (RIP)

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Each Assay Format Has Implications for the Results it Reports

• ELISAs do have several issues related to design, especially non specific background and the need to stick the antigen to a solid surface

• The extensive washes required to reduce background can reduce the ability to detect low affinity antibodies as they can get washed away

• Incubation times may not allow for sufficient binding of low affinity antibodies- too long can lead to dissociation too

Each Assay Format Has Implications for the Results it Reports

• The bridging format appears to have the least background and does not require the use of an anti-human Ig reagent and its associated validation. Care must be taken to avoid loss of low affinity antibodies due to the bispecific nature of the binding.

• RIP assays tend to be the more sensitive assay format as compared to ELISAs*. Not all methods to precipitate complexes, such as Protein A/G, necessarily detect all Ig isotypes and subclasses and the format of choice should be justified by the sponsor

*Swanson, S.J., et. al., (2004) Nephron Clin Practice Vol 96 p88-95

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Surface Plasmon Resonance (SPR)

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SPR Assays - Advantages and Disadvantages

• SPR assays involve the exposure of drug in a lawn of dextran that do not affect the structure of the product to the extent of an ELISA

• It is a real-time procedure and is therefore fast and also detects rapidly dissociating antibodies which can be missed by other methods

• SPR assays do not require extensive wash steps and can not only detect low affinity antibodies but characterize the binding activity

• SPR equipment is expensive and needs expert knowledge to design, run and analyze data if the results are not to be spurious

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Selecting the Assay Cut Point Impacts Reporting Rates

0.000

0.5001 5 9

13

17

21

25

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Sample Number

MeanNSB

Cutpointat 95%negative

X

Op

tic

al D

en

sit

y (

OD

)

NegativeControl

• How the assay cut point is selected dictates when a sample is deemed negative or positive

• The assay cut point thus has impact on the rate of seroconversion reported

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Circulating Therapeutic Can Interfere With Antibody Detection in Antibody Assays

Drug Coat

CirculatingDrug

Serum AntiDrug Antibody

No Binding to Plate

Drug interference can cause significant problems in detection of antibody responses due to the presence of product in samples collected for antibody assessment. This normally results in an artefactually low estimate of antibody content of affected samples and can be so pronounced as to cause false negative results

One has to Understand the Impact of Drug in the Sample if the Screening Assay is to be Meaningful

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.500

0.0

8

0.1

6

0.3

2

0.6

3

1.2

5

2.5

5.0 10

20

40

80

160

320

640

128

0

µg/ml drug added to Serum Sample

Titer 3.1Titer 2.9Titer 2.7Titer 2.5

Limit ofdetection T

itre

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Identification of Immunoglobulin Class

• It may be useful to identify the class and subclass of immunoglobulin detected in a sample but depends on the product and patient responses:

• The disease state or route of administration of drug induces an antibody response of classes other than IgG

• IgA in certain skin autoimmune disorders

• IgE often manifests itself in the patient before being detected in a screening assay.

• Should hypersensitivity be detected in clinical trials, the development of an assay is warranted

• This may be able to detect and avoid reactions to subsequent doses

A Histamine Release Assay to Measure Anti-drug IgE

IgE receptortransfected cell

IgE

IgE loaded cell

Drug

IgE receptorscrosslink

HistamineRelease

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Neutralizing Capacity of Antibodies

•The neutralizing capacity of antibodies to many products should be tested in a biological assay

•Alternative binding-based assays may be appropriate for certain products particularly when used as the potency assay for release– which are often more sensitive than cell based assays

•There is discussion about the utility of neutralizing antibody assays in respect to clinical impact (in vitro tests may not reflect what occurs in vivo).

•From a risk based approach, identification of a neutralizing response can allow for subset analysis of weak signals in the patient population – patients with a neutralizing response may be assessed for PK and PD but this may not be straightforward (e.g. in the oncology setting)

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Bioassays for Neutralizing Antibodies

• Assay responses to product may take the form of cell proliferation or growth inhibition, secretion of protein, gene expression etc.

Cell Response

No Cell Response

Product

Neutralizing Antibody

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A Cytokine Neutralizing Antibody Bioassay

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

1.800

2.000

1 10 100 1000 10000 100000

Abs 4

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Serum Concentration [Dilution]

Cytokine

Serum Pre Exposure

Serum Post Exposure

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Conclusions - Immunogenicity

1. The results of immunogenicity testing rely wholly on the design and execution of the assay used to detect and characterize any immune response

2. It is impossible to compare rates of immunogenicity between biotechnology products unless a head to head clinical immunogenicity study is carried out, thus they should be conducted as such

3. Even in such head to head studies, the rates of immunogenicity are dependent on the assay that the biosimilar company uses and may not reflect the rates seen by the innovator, thus only ‘relative’ assessments of rates can be provided.

Acknowledgements

• Tony Mire-Sluis• Gino Grampp• Andrew Fox• Geoff Eich• Richard Markus

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