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Meenu Wadhwa, PhDCytokines and Growth Factors Section, Biotherapeutics Group,

NIBSC - HPA

United Kingdom

Immunogenicity: What

Do We Know and What

Can We Measure?

Conflict of Interest DisclosureMeenu Wadhwa, PhD

Has no real or apparent

conflicts of interest to report.

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Biotherapeutic Products

• Biotechnology derived products have been

successful for therapy of various diseases

– Hormones, enzymes, coagulation factors, cytokines

– Fusion proteins, monoclonal antibodies

• Despite their benefits, therapeutic proteins

have the potential to induce an immune

response due either to their−Foreign nature if of exogenous origin

or

– Due to recognition as non-self

Immunogenicity Profile of Therapeutics

Product Name Protein Indication

% Patients with

Immune Response

Intron A

Roferon

Pegasys

PegIntron

IFN-α2a Hepatitis C

7

25

9

1

Betaferon

Avonex

Rebif

IFN-β Multiple Sclerosis

25 – 45

2 – 6

12 – 28

Eprex

Neorecormon

Procrit

Aranesp

Epo Anemia Rare

Neupogen

NeulastaG-CSF

Myeloregeneration,

neutropeniaNot detected

Leukine GM-CSFMyeloregeneration,

immunostimulation2 – 95

Proleukin IL-2 Oncology 74

Enbrel TNFR 2-Ig Psoriasis, RA 15

Rituximab Anti-CD20

NHL

CLL

SLE

0

26

Humira Fully human anti-TNFα RA 12

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Unwanted Immunogenicity

Proteins Patients

Alter

PK/PD

Neutralize biologic

effects and compromise

further therapy (factor

VIII, IFNa2a, GM-CSF)

Cross-react with native

protein and induce adverse

symptoms (Epo, MGDF)

Non immunogenic

(G-CSF, IFN-g)

No effect

(growth hormone,

insulin)Immunogenic

(induce antibodies)

Potential Clinical Consequences of Immunogenicity

• Benign, non-significant to serious, life-threatening depending

on the therapeutic

– Efficacy—reduction of the clinical response

– Safety—safety issues can occur even when there is no

loss of efficacy

• Acute consequences

– Infusion reactions, anaphylactic reactions

• Non-acute consequences

– Delayed-type hypersensitivity/immune complexes

– Cross-reactivity with an endogenous counterpart

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Factors Influencing Immunogenicity Product & treatment related

• Protein (structure, primary sequence, novel epitopes,

glycosylation, oxidation, deamidation)

• Product impurities, formulation, aggregation, degradation,

• Properties of the protein eg, immunostimulatory,

replacement therapy, physiologically important?

• Dose, route, frequency of administration and duration of

therapy

Patient related

• Age, gender, genetic make-up, ethnic sensitivity (IFN-α2a

more immunogenic in Chinese vs Caucasian hepatitis

patients 39% vs 14%), immune status, disease

Production Process Complex manufacturing process

− Expression system/vector− Fermentation/ cell culture process− Purification process− Formulation and filling − Drug Product

New manufacturer (copy product)

−Manufacturing and/or purification

process different, no access to know-

how of the process/history of product;

limited to public domain information

Step-Wise Process

Changes

Product likely to have

altered characteristics

Carson KL, Nat Biotechnol. 2005; 23 (9):1045-1048.

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Complexity of Proteins

Biosimilars in the EU

Comparability studies are needed to demonstrate the

similar nature, in terms of quality, safety and efficacy,

of the new similar biological medicinal product and the

reference product authorized in the EU

Emphasis has been given to immunogenicity

Any subtle change introduced in the manufacturing

process of a given product can have enormous

implications for immunogenicity

Unwanted ImmunogenicityCurrent Position

Testing for unwanted immunogenicity is integral to product

development (clinical & post-marketing phase) for ensuring:

– Clinical safety of a biotherapeutic

– Product comparability

– When a biosimilar product is developed

EMA Guidance

• Guideline on Immunogenicity Assessment of Biotechnology-

Derived Therapeutic Proteins EMA/CHMP/BMWP/14327/2006

• Guideline on Immunogenicity Assessment of monoclonal

antibodies (in consultation) EMA/CHMP/BMWP/86289/2010

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LMWH

Revised

IFN-a

EMA Guidelines on Biosimilars

mAbs

IFN-b,

FSH

Recent

Consultation

In Preparation

European Medicines Agency. http://www.ema.europa.eu/

Accessed 23 February 2011.

In revision (quality issues)

Unwanted Immunogenicity

• Prediction of immunogenicity– In silico and T cell methods are promising but

information on the true clinical utility of these

approaches in a prospective manner is lacking

• Determination of immunogenicity– Human clinical data needed—cannot be replaced by

use of animal or in vitro or in silico tools

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Unwanted ImmunogenicityThe Most Challenging Issues

• It is impossible to predict

– The incidence of unwanted immunogenicity

– The characteristics of the immune response

– The clinical consequences & significance of

such immunogenicity

• These require assessment in carefully designed

studies

Immunogenicity Testing: A Tiered Approach

Screening assays: for ‘identification’ of all antitherapeutic

binding antibodies

- Enzyme-linked immunosorbent assays (ELISAs): direct, bridging, other formats

- Radioimmunoprecipitation assays (RIPA)

- Surface plasmon resonance (SPR)

- Other technologies

Confirmatory assays: for confirming antibodies

Neutralization assays: for distinguishing neutralizing &

non-neutralizing antibodies.

- Cell-based assay

- Non-cell-based ligand binding assay

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Strategy for Assessment of ImmunogenicityPatient samples taken at appropriate

time-points

Screening Assays-ve samples rejected +ve samples

Confirmatory Assays

Bioassay Confirmed +ve samples Characterization

Assess correlation of characterized antibodies

with clinical responses to biologic therapeutic

Assays for clinical markers and assessment

of clinical response in patients

Immunogenicity Assessment Strategy Design and Interpretation

• Studies need to be carefully and prospectively designed to ensure all procedures are in place prior to initiation

– Selection, assessment, characterization and validation of assays

– Identification of appropriate sampling points, duration of testing

– Sample volumes and sample processing/storage

– Selection of statistical methods for analysis of data

• This applies to all assays as shown in strategy slide

• Strategy needs to be established on a case-by-case basis –product, patients, expected clinical parameters

– In chronic use – sequential sampling for a year

– In view of variability of antibody responses, adequate numbers of patients needed

• However, unwanted immunogenicity may occur at a level, which is not detected in studies pre-approval so assessment post-approval, as part of pharmacovigilance surveillance is needed

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Comparative Immunogenicity

• Studies need to be designed to demonstrate whether the immunogenicity of the products is the same or significantly different

• This may affect the design of the studies & their interpretation

• A homogeneous and clinically relevant patient

population should be selected. Head-to-head studies

using same assays & sampling strategy needed

• The consequences of immunogenicity must also be compared

• Post-approval assessment may be necessary, usually as part of pharmacovigilance surveillance

Immunogenicity of Biosimilars

The immunogenicity of the marketed product does not influence the need for comparative immunogenicity studies

However, if the imunogenicity profiles of marketed and biosimilar products are significantly different, they may be considered DISSIMILAR

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Antibody Frequency for Biosimilars (Presubmission Studies)

Biosimilar Ab Incidence Reference Ab Incidence

Omnitrope (SC) 0/51 (0.0%) Genotropin 1/44 (2.3%)

Valtropin (SC) 3/98 (3.4%) Humatrope 1/49 (2.0%)

Binocrit (IV) 2/314 (0.6%) * Erypo 3/164 (1.8%) *

Silapo (IV)

Silapo (SC)

0/305 (0.0%)

0/323 (0.0%)

Erypo

Erypo

0/304 (0.0%)

0/230 (0.0%)

Ratiograstim (SC) 7/356 (2.0%) Neupogen 2/134 (1.5%)

Zarzio (IV / SC)

(phase 1, crossover)

0% Neupogen 0%

Nivestim 3/183 (1.6%) Neupogen 0/95 (0.0%)

Alpheon (SC) § † 31/111 (27.9%) ** Roferon-A 38/99 (38.4%) **

* Prevalence; ** at end of treatment; § Application refused or withdrawn;

† Assay validation insufficient

Courtesy Martina Weise (Bfarm)

Antibodies and Adverse Effects—EPO

Pure red-cell aplasia (PRCA) and anti-EPO antibodies in patients treated with EPO (EPREX)

• 2002 - 13 cases in chronic renal failure patients, rapid development of severe transfusion dependence within months of therapy, resistant to other EPO products

• Pre 1998 – 2/3 cases

•1998 to June’05 – 260+cases worldwide

Casadevall N, et al. N Engl J Med. 2002;346(7):469-475.

Safety Study for Binocrit Suspended

− No increased immunogenicity from

IV use in patients with renal anaemia

or SC use in cancer patients (both

licensed)

− Postmarketing SC trial in previously

untreated renal anaemia patients: two

cases of neutralizing Ab

Binocrit approved - 2007

Rigorous physico-chemical, biological

characterization & clinical trial data Brockmeyer C, et al. Eur J Hosp Pharm Prac. 2009;15(2):34-40.

>Sixty PRCA cases identified in Thailand. Fourteen EPO products marketed. Link to product(s)?

Cause(s) ?

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Immunogenicity of Other Products

Product Type Disease

Antibody

Incidence %

Remicade Anti-TNFCrohn

RA

61

8

Rituxan Anti-CD20

Non-Hodgkin

SLE

CLL

0

65

Campath Anti-CD52 B-CLL

RA

1.9

29-63

• IFN-β - Incidence of neutralizing antibodies varies –

– Avonex 2% to 6%; Rebif 12% to 28%; Betaferon 25% to 45%

– Loss of efficacy; abs cross-reactive and impact on

disease management and QOL

• Monoclonal antibodies – Ab incidence dependent on product,

disease, age; loss of efficacy noted in some cases.

Immunogenicity Guideline

• Recommendations for routine monitoring of changes in

clinical response and linking immunologic findings to

clinical events

– Immunogenicity as part of all clinical trials (→ indication

specific differences possible)

– Evaluate all patients (… and not in a symptom-driven manner)

– Standardize sampling schedule as much as possible

• Specifically analyse adverse events for linkage to an

unwanted immune response (ie, also in a symptom driven-

manner)

• Provide guidance on how prescriber should handle patient

in case unwanted immune response occurs (increase

dose/discontinue, etc)

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Biosimilars: Unwanted Immunogenicity

Quote from EMA BMWP chairman:

‘Unwanted Immunogenicity is the biggest

challenge for the approval of Biosimilars’