The document discusses immunogenicity as a key issue for biotechnology-derived products. It summarizes guidelines for evaluating the immunogenic potential of therapeutic proteins. Immunogenicity can negatively impact efficacy and safety through the production of antibodies, including neutralizing antibodies. The guidelines provide recommendations on non-clinical and clinical assessment of immunogenicity and strategies for risk management. Evaluation of immunogenicity is important both before and after market approval.

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Immunogenicity as a key
issue for biotechnology-
derived products.

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Carlo Pini
(carlo.pini@iss.it)
Director
National Center for Immunobiologicals Research and
Evaluation - CRIVIB
Istituto Superiore di Sanità
Roma
Italian Delegate - Biologics Working Party (CHMP/BWP)
EMA - London

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Disclaimer
• The content of the following presentation represents
the speaker’s view and does not reflect any official
point of view.
• According to the EMA policy (0044 MA/513078/2010)
– No direct conflict of interest
– One indirect conflict of interest

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Biologicals/biotechnologicals products
• Biologicals/biotechnologicals products are
complex macromolecules which cannot be fully
characterised from the analytical point of view but
whose quality attribute are also largely defined
by the manufacturing process.
• They may be:
– copies of naturally occurring molecules normally
present in the human body such as interferons, growth
factors (G-CSF, erithropoietin ) antibodies, etc.
– “foreign molecules” which have been designed to
interfere with a certain pathogenic mechanism.
– Molecules which have been intentionally designed to
be immunogenic (recombinant vaccines).
Immunogenicity is therefore a positive characteristic
of the product.

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Immunogenicity
• With the exception of vaccines, immunogenicity is a
negative unwanted property of a given molecule, and a
number of attempts have been made to design a recombinant
molecule to reduce/abolish this characteristic.
• The predictive value of non-clinical studies for evaluation of
immunogenicity of a biological medicinal product in
humans is low due to inevitable immunogenicity of human
proteins in animals.
• Differences between neutralising vs non-neutralising
antibodies.
• In the clinical setting, careful planning of immunogenicity
evaluation should include data systematically collected from
a sufficient number of patients.
• Immunogenicity issues should be further addressed in the
Risk Management Plan.

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Immunogenicity
• The consequences of an immune reaction to a therapeutic protein range
from transient appearance of antibodies without any clinical
significance to severe life-threatening condition.
• The immunogenicity of therapeutic proteins. can be considered to be
patient-, disease- or product-related
• data on possible unwanted immune reactions to therapeutic proteins are
required before marketing authorisation, problems may still be
encountered in the post-authorisation period.
• Depending on the immunogenic potential of the therapeutic protein and
the rarity of the disease, the extent of immunogenicity data before
approval might be limited
• Further systematic immunogenicity testing might become necessary
after marketing authorization, and may be included in the risk
management plan.

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Immunogenicity
• A lot of experience has been gained since the
very first recombinant product (murine
monoclonal antibody – Muromomab CD3 –
middle 1980s) was authorized.
• As a consequence, several technical approaches
have been introduced to manufacture less and
less immunogenic products
• For example, murine MoAb from ascites have
been replaced by fully humanized MoAbs where
the only “foreign” structure is represented by a
few aas defining the binding region.

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Immunogenicity
• The progress of the scientific knowledge in the field has suggested
in the recent past that the immunogenicity issue should be largely
treated in a CHMP Guideline (CHMP/BMWP/14327/06).
• The Guideline came into effect in April 2008
• A number of important aspects have been covered in the document
• The scope of the Guideline is not restricted to recombiant products
but it applies to biologicals in general, when feasible.
• Very important point, this guideline should be considered together
with the relevant guidelines on Biosimilar products and on
Comparability (quality, non clinical and clinical parts).
– Impact of changes in the manufacturing process on immunogeniciy
– Immunogenicity of the biosimilar product
• Therefore evaluation of immunogenicity should be a
multidisciplinary task, encompassing joint efforts of quality, non-
clinical and clinical experts.

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Immunogenicity (CHMP/BMWP/14327/06)
• Guideline on immunogenicity assessment of Therapeutic proteins
– Immunogenicity guideline: general section
• Basic concepts, immunogenicity and humanisation, molecular size, percentage
of murine parts, etc.
• Patients and disease related risk factor.
• In the comparability exercise (post changes products and biosimilars, the
immunogenicity in non clinical model may provide useful information.
The antibody you get may be irrelevant or may neutralise the whole activity of
the drug. Distinction between neutralising and non-neutralising antibodies.
PK influencing antibodies not easily determined and distinguished from the
neutralising antibody.
• Difference between infusion reaction and anafilactic reaction which should
prevent the patient to get treated.
• Risk management and standardised measurements methods.
– Immunogenicity guideline: Annex I
• Unwanted immunogenicity and various types of antibodies and different
antibodies require different methods.
• It is impossible to predict a number of factors with antibodies and studies
are required.

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General section
• Factors that may influence the development of an
immune response against a therapeutic protein
– Patient- and disease-related factors
• Genetic factors modulating the immune response
• Disease-related factors and age
• Concomitant treatment
• Duration, route of administration, treatment modalities
• Previous exposure to similar or related proteins
– Product related risk factors of immunogenicity
• Protein structure
• Formulation
• Aggregates and impurities

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General section
• Non-clinical assessment of immunogenicity and its
consequences
– Therapeutic proteins show species differences in most cases. Thus,
human proteins will be recognised as foreign proteins by animals.
– Measurement of antibodies in non-clinical studies are however
requested as part of repeated dose toxicity studies (see ICH S6)
– the comparison of the antibody response to the reference product in
an animal model may be part of the comparability exercise both for
similar biological medicinal products and for changes in
manufacturing processes
– An immune response to a therapeutic protein representing a
counterpart to an endogenous protein may result in cross-reactivity,
directed to the endogenous protein in cases where endogenous
protein is still produced.
– Both humoral and cellular immune responses (where relevant)
should be considered.
– Animal homologous models useful

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General section
• Assays for detecting and measuring immune
responses in humans.
– Assay strategy
– Antibody assay
• Screening
• Confirmation
• Specificity
• Neutralisation assay
• Assay validation, standardisation and controls
• Characterisation and strategy

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Consequences of immunogenicity
• Efficacy
– Factors inducing clinical consequences can be the
epitope recognised, affinity, class of the antibody, the
amount of antibodies generated, etc.
– Capability of being cytotoxic (cell mediated,
complement mediated)
– Antibodies recognising epitopes on the therapeutic
protein not linked to activity are expected to be
associated with less clinical consequences.
– “Neutralising” antibodies, interfering with biological
activity by binding to or near the active site, or by
induction of conformational changes, can induce loss
of efficacy.

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Consequences of immunogenicity
• Safety
– patients who develop antibodies are more likely to
show infusion-related reaction
– the consequence of immunogenicity is product-
specific and can elicit unexpected clinical
symptoms.
– Immediate hypersensitivity
– Delayed hypersesitivity and immune complexes
– Cross-reactivity with an endogenous counterpart

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Consequences of immunogenicity
• Immunogenicity and Clinical Development
– Immunogenicity assessment should be part of the
clinical trials, since the correlation to clinical efficacy
and safety is important
– Several factors such as dose, schedule and treatment
modalities influence the development of an immune
response against a therapeutic protein
– For products intended for chronic use, it may be
necessary to study the evolution and persistence of an
observed immune response

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Consequences of immunogenicity
• Consequences on pharmacokinetics of the
product
– Influence of non-neutralising antibodies
– Methodology aspects to assess comparability of
immunogenicity potential as part of a
comparability exercise
– Applicants should make an effort to select a
homogeneous and clinically relevant patient
population that allows for such comparisons.

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Risk Management Plan (RMP)
• The applicant should present a risk management plan in
accordance with current EU legislation and CHMP
Guideline on Risk Management Systems for Medicinal
Products for Human Use (EMEA/CHMP/96268/2005).
• The extent of data on immunogenicity that can be obtained
during the clinical development programme of a
biotechnology-derived product before approval depends on
the event rate, driven both by the immunogenic potential of
the protein and the rarity of the disease.
• Therefore, further systematic immunogenicity testing might
become necessary after marketing authorization, and may be
included in the Risk Management Plan.
• For planning immunogenicity assessment in the post
marketing setting, the same recommendations apply as
discussed in previous sections of this guideline.

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Annex 1
• Further details on methods for assessment and
characterisation of immunogenicity
– Types of antibody assays
• Screening assays
• Assays for confirming antibody positivity
• Assays for dissecting the specificity
• Neutralization assays
• Assays for assessing cell-mediated immune responses
• Assay characteristics
• Standardisation, reference materials, well characterized
controls and assay validation, interpretation of Results

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Immunogenicity of biologicals

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Product-specific guideline

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Immunogenicity of MoAbs
• This guideline addresses issues relating to the
unwanted immunogenicity of monoclonal
antibodies (mAbs) intended for clinical use.
• These include factors impacting on:
– immunogenicity of mAbs,
– clinical consequences of immunogenicity
– assay related problems, assessing neutralizing
antibodies induced by mAbs
– consideration of a risk-based approach for the
evaluation of immunogenicity of mAbs.

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Specific Issues
• While many aspects of immunogenicity of mAbs
are not different from those for other therapeutic
proteins, there are several aspects that require
more specific considerations.
• some specific aspects of immunogenicity are
exclusively or primarily relevant for mAbs or
novel mAb derivatives (e.g. Fab fragments, scFv,
nanobodies, minibodies)
• This guideline considers the major quality and
clinical aspects that are important for adequately
addressing the immunogenicity of MoAbs

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Antibodies anti-MoAbs
• Detection of antibodies against mAbs is often
more problematic, difficult and can be technically
challenging.
– Presence of mAb product in samples for analysis
– Confirmatory Assays
– Controls
• Assessing the neutralising capacity of antibodies
induced against mAbs
– Mechanism of action of the MoAb (Complement, cells)
• Immunogenicity risk management of mAbs

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Risk identification
• Prior knowledge
• Mab structure
– the immune response is predominantly anti-
idiotypic (as the complementarity determining
regions - CDR - are hypervariable in sequence
– Hidden epitopes and new epitopes in conjugated
MoAbs
– Impurities and other quality attributes
• Mechanism of action
• Clinical factors (age, previous exposure, etc.)

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Conclusions
• Immunogenicity of biologicals is a well know
phenomenon
• However mechanisms involved are very complex and
deserve specific investigations and scientific
approaches
• The nature of the induced immune response needs to
be clarified and it impact on safety and efficacy should
be understood (neutralising vs non-neutralising
antibodies)
• Trials available at the time of the MAA may not be
large enough to identify less frequent patients capable
of mounting an immune response against the product.
• Risk management Plan must take into account all
potential issues linked with immunogenicity.