“Biotherapeutic Medicines – regulatory challenges and currentpractices. Approaches for harmonization” Moscow, Russia May 16,2013. IFPMA/AIPMImmunogenicity testing of BiotechnologyProducts and the Impact to BiosimilarsBarry CherneyExecutive Director Product QualityAmgen Inc
2• Administration of non native or even ‘humanized’proteins to either animals or humans can eliciteither an antibody response, cellular response, orboth if the immune system recognizes the proteinas foreign.• All protein products have some level ofimmunogenicity, with varying impact to patients.• Although there are multiple theoretical causes ofimmunogenicity, very few real examples of why aproduct is immunogenic have been publishedProtein Products have ImmunogenicPotential
3Generalized Protein DomainsReceptorbinding/functional domainNon-receptorbinding/functional domain
4Effect of antibodies on the function ofproteinsCell Response NeutralizingantibodiesNon-neutralizingantibodiesNo cellresponse
5Clinical Concerns in Testing forAntibodies - Effects on PK/PD• The binding of antibodies to product has beenshown to potentially affect (by extension orreduction) the half life in blood through theinfluence on clearance mechanisms.• Biodistribution of product has also beenshown to be affected, such as lack oftargeting to skin or tumour sites.• If product is cleared differently and itsdistribution is different, then its ability to havethe desired biological effect may also bealtered.
6Clinical Concerns in Testing forAntibodies - Effects on Efficacy• Antibodies that alter the PK/PD of theproduct may have an effect on its efficacy. Ifthe drug remains longer in the circulation,efficacy can be enhanced. If half life isreduced, so potentially, is its efficacy.• The presence of neutralizing antibodies candirectly inhibit the biological activity of theproduct and thus clinical efficacy may bereduced or abrogated.
7Clinical Concerns in Testing forAntibodies - Effects on Safety• Extending the half life of a product caninfluence its toxic properties.• Redistributing a product to different sites maypotentially also have safety implications.• The presence of complexes of product andantibody can have physiologicalconsequences.
8• Immune responses to product can lead to:• Anaphylaxis• Injection site reactions• Flu like syndromes• Allergic responses• One of the most serious adverse eventsoccurs when neutralizing antibodies toproduct cross react with endogenous proteinsthat have a unique physiological role.Clinical Concerns in Testing forAntibodies - Effects on Safety
• “Comparative assessment of unwanted immune responses againstthe biosimilar and the reference mAb are normally undertaken aspart of the clinical study”*• “A risk-based approach can provide a starting point from which thefurther concept of immunogenicity testing can be designed, but dueto the diversity of risk factors, as discussed in this guideline, and thevariety of mAbs and mAb-related products, the recommendationsgiven here cannot be generalized.”**• “Assessment is based on the identification of risk factors inherent tothe particular mAb in question, the final drug product and the treatedpatient population. The mechanism of action and the basic structure(chimaeric, humanized, fully human) are not sufficient for decidingon the attribution of risk level. For a risk-based approach, applicantsneed to define what “risk” in this context means.”*** Guideline on similar biological medicinal products containing monoclonal antibodies – non clinicaland clinical issues CHMP 2010 ** Guideline on immunogenicity assessment of monoclonalantibodies intended for in vivo clinical use. CHMP Nov. 2010EMA mAb biosimilar and immunogenicityguidelines on the “risk-based approach”9
10• “At least one clinical study that includes acomparison of the immunogenicity of the proposedproduct to that of the reference product willgenerally be expected “• “The extent and timing …..of the clinicalimmunogenicity program will vary depending on arange of factors including the extent of analyticalsimilarity and the incidence and clinicalconsequences of immune response for thereference product. “*Scientific Considerations in Demonstrating Biosimilarity to a Reference ProductFDA. Feb 2012FDA’s draft biosimilar guideline* alsoincludes “a risk based immunogenicityapproach”
Risk Based Approaches toImmunogenicity TestingRisk = Probabilityharm x Severityharm• Severity outweighs the probability of a riskoccurring.How many patients arelikely to mount an immuneresponse?What happens to the patientif they mount an immuneresponse?11
Considerations in Assessing Risk ofImmunogenicity - Probability AnalysisLikely Lower Probability Likely Greater ProbabilityImmunosupressed patients Autoimmune diseaseSingle dosing Chronic dosingMore ‘Human’ ‘Foreign’IV administration SubcutaneousHighly pure ImpureNo aggregates Aggregates12Risk = Probabilityharm x Severityharm
13Considerations in Assessing Risk ofImmunogenicity - Severity AnalysisLikely More Severe Likely Less SevereEndogenous version No endogenous versionUnique activity Redundant activitySole therapy Other therapiesLife threatening disease Non life threatening diseaseChronic disease End stage diseaseNon reversible AE Reversible AEReplacement therapy Non replacement therapyAnaphylactic response Non anaphylactic responseRisk = Probabilityharm x Severityharm
Examples of Anti Drug Antibodies: Incidenceand Clinical Impact14Rate ofAntibodiesMoleculeAntibodyIncidenceClinical ImpactHighr-Human alpha-galactosidase88% none reportedHighr-chimericanti-TNFa10-57%HypersensitivityPK affectedefficacy unchangedModerater-chimericanti-GPIIb/IIIaFab7-19%Higher incidence afterre-administration,TCP higher in ab+patientsModerater-HumanGlucocere-brosidase13%NeutralizingantibodiesrareLowr-HumanThrombopoietintruncated, PEG0.5-4%neutralizingantibodiesTCPLowr-Human TissuePlasminogenActivator<1% none reported
Considerations for Analytical Testing Strategies based on theoutcome of the Risk Assessment ScoreExamples of High scoreconsiderationsExamples of Low scoreconsiderationsTest samples in real time Batch test samples at the end ofthe studyMore sensitive assays early indevelopmentDevelop increasingly sensitiveassays during developmentHighly conservative approachto setting cut pointsLess conservative approach tosetting cut pointsTest positive binding samplesfor neutralization in real timeBatch positive binding samplesafter initial screening and thentest for neutralizationNeed to develop sensitiveneutralization assaysLess need for very sensitiveneutralization assays15
16Biosimilar Specific Issues withImmunogenicity Testing• The rate of immunogenicity detected in an assay is whollydependent on the assay chosen, its controls, executionand the clinical study sample plan design• Biosimilar companies are unlikely to know the details ofthe 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 AssayAn Optimal screening assay should:• Be sensitive enough to detect any level ofspecific antibody present in patient sera.• Have no false negatives• Have a few false positives able to beproved false positives• Discriminate between pre-existingantibodies and treatment inducedantibodies• Detect antibodies in the presence of drug• Be reproducible17
18The Choice of Assay Format can Heavily Influencethe Rate of Immunogenicity Results ReportedEnzymeColouredreagentClearreagentDetectingantibodyAnti-drugantibodyDrugSerumAntibodyConjugatedDrugDrugDetectionSystemAdd SerumSamplePrecipitate antibodybound drugMeasureradioactivityin pelletELISABridgingELISARadioImmunoprecipitationAssay (RIP)
19Each Assay Format Has Implications for theResults it Reports• ELISAs do have several issues related to design,especially non specific background and the need tostick the antigen to a solid surface• The extensive washes required to reduce backgroundcan reduce the ability to detect low affinity antibodiesas they can get washed away• Incubation times may not allow for sufficient binding oflow affinity antibodies- too long can lead todissociation too
Each Assay Format Has Implications forthe Results it Reports• The bridging format appears to have the leastbackground and does not require the use of an anti-human Ig reagent and its associated validation. Caremust be taken to avoid loss of low affinity antibodiesdue to the bispecific nature of the binding.• RIP assays tend to be the more sensitive assayformat as compared to ELISAs*. Not all methods toprecipitate complexes, such as Protein A/G,necessarily detect all Ig isotypes and subclasses andthe format of choice should be justified by the sponsor*Swanson, S.J., et. al., (2004) Nephron Clin Practice Vol 96 p88-9520
22SPR Assays - Advantages andDisadvantages• SPR assays involve the exposure of drug in a lawn ofdextran that do not affect the structure of the productto the extent of an ELISA• It is a real-time procedure and is therefore fast andalso detects rapidly dissociating antibodies which canbe missed by other methods• SPR assays do not require extensive wash steps andcan not only detect low affinity antibodies butcharacterize the binding activity• SPR equipment is expensive and needs expertknowledge to design, run and analyze data if theresults are not to be spurious
23Selecting the Assay Cut Point ImpactsReporting Rates0.0000.500 1591317212529Sample NumberMeanNSBCutpointat 95%negativeXOpticalDensity(OD)NegativeControl• How the assay cut point is selected dictates when asample is deemed negative or positive• The assay cut point thus has impact on the rate ofseroconversion reported
24Circulating Therapeutic Can Interfere WithAntibody Detection in Antibody AssaysDrug CoatCirculatingDrugSerum AntiDrug AntibodyNo Binding to PlateDrug interference can cause significant problems in detection of antibody responses due tothe presence of product in samples collected for antibody assessment. This normallyresults in an artefactually low estimate of antibody content of affected samples and can beso pronounced as to cause false negative results
One has to Understand the Impact of Drug in theSample if the Screening Assay is to be Meaningful0.000.501.001.502.002.503.003.50 00.080.160.320.631.252.55.0102040801603206401280µg/ml drug added to Serum SampleTiter 3.1Titer 2.9Titer 2.7Titer 2.5Limit ofdetectionTitre25
26Identification of Immunoglobulin Class• It may be useful to identify the class and subclass ofimmunoglobulin detected in a sample but dependson the product and patient responses:• The disease state or route of administration ofdrug induces an antibody response of classesother than IgG• IgA in certain skin autoimmune disorders• IgE often manifests itself in the patient beforebeing 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 tosubsequent doses
A Histamine Release Assay to MeasureAnti-drug IgEIgE receptortransfected cellIgEIgE loaded cellDrugIgE receptorscrosslinkHistamineRelease27
Neutralizing Capacity of Antibodies•The neutralizing capacity of antibodies to many productsshould be tested in a biological assay•Alternative binding-based assays may be appropriate forcertain products particularly when used as the potencyassay for release– which are often more sensitive than cellbased assays•There is discussion about the utility of neutralizingantibody assays in respect to clinical impact (in vitro testsmay not reflect what occurs in vivo).•From a risk based approach, identification of a neutralizingresponse can allow for subset analysis of weak signals in thepatient population – patients with a neutralizing response may beassessed for PK and PD but this may not be straightforward (e.g. inthe oncology setting)28
29Bioassays for Neutralizing Antibodies• Assay responses toproduct may takethe form of cellproliferation orgrowth inhibition,secretion of protein,gene expressionetc.Cell ResponseNo CellResponseProductNeutralizing Antibody
30A Cytokine Neutralizing Antibody Bioassay0.0000.2000.4000.6000.8001.0001.2001.4001.6001.8002.0001 10 100 1000 10000 100000Abs450Serum Concentration [Dilution]CytokineSerum Pre ExposureSerum Post Exposure
31Conclusions - Immunogenicity1. The results of immunogenicity testing rely wholly on the designand execution of the assay used to detect and characterizeany immune response2. It is impossible to compare rates of immunogenicity betweenbiotechnology products unless a head to head clinicalimmunogenicity study is carried out, thus they should beconducted as such3. Even in such head to head studies, the rates ofimmunogenicity are dependent on the assay that the biosimilarcompany uses and may not reflect the rates seen by theinnovator, thus only ‘relative’ assessments of rates can beprovided.
Acknowledgements• Tony Mire-Sluis• Gino Grampp• Andrew Fox• Geoff Eich• Richard Markus32