mAbs

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/kmab20

Proceedings of the 14th European immunogenicity

platform open symposium on immunogenicity of
biopharmaceuticals

Sophie Tourdot, Daniel Baltrunkonis, Sofie Denies, Viswanath Devanarayan,

Joanna Grudzinska-Goebel, Arno Kromminga, Gregor P. Lotz, Laurent
Malherbe, Lydia Michaut, Karin N. Weldingh, Joao A. Pedras-Vasconcelos,
Laura. I. Salazar-Fontana, Sebastian Spindeldreher, Zuben Sauna, Veerle
Snoeck, Daniela Verthelyi & Daniel Kramer

To cite this article: Sophie Tourdot, Daniel Baltrunkonis, Sofie Denies, Viswanath
Devanarayan, Joanna Grudzinska-Goebel, Arno Kromminga, Gregor P. Lotz, Laurent
Malherbe, Lydia Michaut, Karin N. Weldingh, Joao A. Pedras-Vasconcelos, Laura. I. Salazar-
Fontana, Sebastian Spindeldreher, Zuben Sauna, Veerle Snoeck, Daniela Verthelyi &
Daniel Kramer (2024) Proceedings of the 14th European immunogenicity platform
open symposium on immunogenicity of biopharmaceuticals, mAbs, 16:1, 2324801, DOI:
10.1080/19420862.2024.2324801

To link to this article: https://doi.org/10.1080/19420862.2024.2324801

© 2024 The Author(s). Published with

license by Taylor & Francis Group, LLC.

Published online: 05 Mar 2024.

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MABS
2024, VOL. 16, NO. 1, 2324801
https://doi.org/10.1080/19420862.2024.2324801

MEETING REPORT

Proceedings of the 14th European immunogenicity platform open symposium on

immunogenicity of biopharmaceuticals
Sophie Tourdota, Daniel Baltrunkonisb, Sofie Deniesc, Viswanath Devanarayand, Joanna Grudzinska-Goebele,
Arno Krommingaf, Gregor P. Lotzg, Laurent Malherbeh, Lydia Michauti, Karin N. Weldinghj, Joao A. Pedras-Vasconcelosk,
Laura. I. Salazar-Fontanal, Sebastian Spindeldreherm, Zuben Saunan, Veerle Snoecko, Daniela Verthelyip,
and Daniel Kramerq
a
Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc, Andover, MA, USA; bResearch and Development, Clinical Pharmacology and Bioanalytics,
Clinical Bioanalytics, Groton, CT, USA; cSD Analytics, Bellem, Belgium; dEisai Inc, Nutley, NJ, USA; eDMPK Project Management, Pharmaceuticals R&D,
Bayer AG, Berlin, Germany; fBioNTech SE, Mainz, Germany; gRoche Pharma Research and Early Development (pRED), Roche Innovation Center
Munich, Hoffmann-La Roche Ltd, Penzberg, Germany; hLilly Research Laboratories, a Division of Eli Lilly and Company, Indianapolis, IN, USA; iNovartis
Biomedical research, PK Sciences, Basel, Switzerland; jDepartment of Clinical Immunogenicity Analysis, Novo Nordisk A/S, Maaloev, Denmark;
k
Division of Biotech Review and Research III, Office of Biotechnology Products, Center for Drug Evaluation and Research, US Food and Drug
Administration, Silver Spring, MD, USA; lLAIZ Regulatory Science, Lausanne, Switzerland; mIntegrated Biologix GmbH, Basel, Switzerland; nDivision of
hemostasis, Office of Plasma Protein Therapeutics; Office of Therapeutic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug
Administration, Silver Spring, MD, USA; oTranslational Biomarkers and Bioanalysis, UCB Biopharma SRL, Braine-l’Alleud, Belgium; pDivision of
Biologics Research and Review III; Ofrfice of Biotechnology Products; Center for Drug Evaluation and Research; Office of Pharmaceutical Quality, US
Food and Drug Administration, Silver Spring, MD, USA; qGlobal Scientific Advisor Immunogenicity, Translational Medicine & Early Development,
Sanofi Aventis Deutschland GmbH, Frankfurt am Main, Germany

ABSTRACT ARTICLE HISTORY

Biologics have revolutionized disease management in many therapeutic areas by addressing unmet Received 30 January 2024
medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, Revised 11 February 2024
the development of unwanted immune responses directed against these drugs, humoral and/or cellular, Accepted 13 February 2024
can hinder their efficacy and have safety consequences with various degrees of severity. Health autho­ KEYWORDS
rities ask that a thorough immunogenicity risk assessment be conducted during drug development to Anti-drug antibody; clinical
incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversifica­ relevance; immunogenicity;
tion and complexification of biologics, which today include modalities such as multi-domain antibodies, risk assessment
cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of
establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The
European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals
and its one-day training course gives experts and newcomers across academia, industry, and regulatory
agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we
report the discussions that took place at the EIP’s 14th Symposium, held in April 2023. The topics covered
included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assess­
ment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated
with new modalities, which were discussed in a dedicated session.

Introduction
The European Immunogenicity Platform’s 14th Open
Symposium on Immunogenicity of Biopharmaceuticals
was held April 26–28, 2023 in Lisbon, Portugal. The long­
evity of this annual meeting highlights how the develop­
ment of unwanted immune responses remains a hindrance
to providing patients with safe and efficacious biologic-
based treatment and still needs mitigation solutions. The
emergence of novel modalities such as multidomain, multi­
specific, conjugated antibodies, novel scaffolds, alongside
cell and gene therapy products, including those for gene
editing has brought additional complexity to immunogeni­
city risk assessment. In the absence of specific regulatory
agency guidelines for some of these biologics, such as
mRNA-LNP products, sharing immunogenicity risk and
mitigation approaches and practices amongst developers is
paramount to acceleration of drug development. In this
context, the Symposium brought together experts in the
various aspects of immunogenicity risk assessment, includ­
ing assay development and analysis for clinical immuno­
genicity assessment or pre-clinical mitigation by design,
accompanied by regulatory perspectives on current and
future approaches in these two areas. In addition to the
plenary sessions, a training day with its “Bring your own
problems” sessions offered participants an opportunity to
brainstorm ahead of the meeting, on specific issues they
might have encountered. Altogether, the meeting was
a testimony of the highly collaborative spirit of the immu­
nogenicity community. The topics and discussions are pre­
sented in this report.

CONTACT Sophie Tourdot sophie.tourdot@pfizer.com Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc, 1 Burtt Road, Andover, MA 0810, USA
© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the
posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
2 S. TOURDOT ET AL.
Immunogenicity monitoring and clinical relevance
Given the potential consequences of the development of an
unwanted immune response, confident monitoring of immu­
nogenicity in the clinic is critical to allow adequate mitigation.
Assessment of humoral responses, i.e., measurement of anti-
drug antibodies (ADA), and interpretation of the results
require the establishment of qualified/validated assays.
Recent advances in the field, such as the use of singlicates or
moving toward replacing ADA titers by signal-to-noise (S/N),
were presented and covered at the “Bring your own problems”
round table discussion on training day
Signal-to-noise in ADA measurement
Dr Viswanath Devanarayan (Eisai Inc., USA) discussed the use
of the S/N approach as an alternative to ADA titers. This
presentation started initially with a review of key findings
from Manning et al.1 that included data from several immu­
nogenicity assays and clinical studies from different pharma­
ceutical companies, followed by a deeper dive into specific
assays and studies to provide more insights on the S/N versus
titer to quasi-quantify ADA levels. Eleven of the 15 ADA
assays had >0.8 Spearman correlation between S/N versus
titer, and three other assays had correlations between 0.6 and
0.8. Except for one of the assays, both S/N and titer had
comparable levels of correlation to pharmacokinetics (PK) (p
> 0.05, Hittmer’s test). In addition, the ADA kinetics were
similar with respect to both S/N and titer. A closer look at
one of the assays (A7) that had dosing information available
with a wide range of ADA magnitude revealed that the impact
of ADA on PK is similar between S/N and titer, with greater
variability in the first quartile of titers. Results from 500 simu­
lations of the data based on this assay revealed that
a correlation greater than 0.8 between S/N versus titer should
yield a similar conclusion about the clinical impact in terms of
their association with PK and pharmacodynamics (PD). One
of the assays (A1) with moderate to high S/N values for several
samples had titer results plateauing at the lower end. Further
evaluation of these samples revealed the presence of preexist­
ing antibodies. The low titer results of these samples could be
due to the low affinity/avidity ADA that gets disassociated
faster or blocked by the exogenous matrix added during titra­
tion. Assay A4 provided a contrasting scenario, with the S/N
plateau at the top for samples with higher titer results. When
assessing S/N and titer association to PD via quartiles, the
plateau effect of S/N due to the low range of enzyme-linked
immunosorbent assay (ELISA) did not affect the ADA impact
assessment on PD; on the contrary, S/N had a more consistent
trend versus PD and with less variability. A review of some of
the assays also revealed that the S/N approach was more
sensitive for identifying treatment-boosted ADA than the
titer approach. The precision of S/N was considerably better
compared to titer, with the %CV across all assays ranging from
9% to 24% for S/N, and 27% to 64% for titer. In conclusion,
some of the key strengths of the S/N approach are that it is
simpler and faster, saving considerable resources (less reagent
use, sample volume, and analyst time), more precise, with
improved differentiation of low-level ADA, and more robust
to low affinity/avidity, whereas the advantages of titer are that
they are better understood and has no assay saturation. While
S/N may suffer from range limitations usually with ELISAs, the
titer approach does not come with the benefits of S/N and is
historically not validated with the same rigor as screening
assays. The S/N approach can be justified during assay valida­
tion by assessing the factors impacting S/N and titer, such as
assay range, precision, drug/target interference, and low affi­
nity/avidity.
Mr. Daniel Baltrukonis (Pfizer, USA) provided an industry
perspective by building upon the publication by Manning
et al.1 ADA data across 17 clinical programs spanning 5 for­
mats/modalities (standard monoclonal antibody (mAb)),
fusion proteins, bispecifics, antibody-drug conjugates, and
gene therapies (anti-adeno associated virus) and both electro­
luminescence (ECL) and ELISA bridging formats were shared.
Strong correlation of r ≥ 0.713 was observed across 14 of the
clinical programs. There were 3 programs in which no correla­
tion between S/N and titer was initially observed (r ≤ 0.077),
but the lack of correlations could be explained by limitations in
the data sets (e.g., size, post-dose mature responses) and lim­
itations of dynamic range in ELISA methodologies.
A comprehensive case study examining the correlation,
kinetics of the ADA response, and clinical impact on PK and
PD using S/N was discussed.2 Concerns with S/N include the
limitation of the assay dynamic range and potential to observe
a prozone effect. In the case of bococizumab, prozone effects
were observed with the bridging ECL assay, but these observa­
tions were in the upper 10% of maximal observed ADA
responses and did not affect clinical interpretation. The same
ADA magnitude-dependent impact on PK and PD was
observed using either titer or S/N.2
Dr João Pedras-Vasconcelos (Food and Drug
Administration (FDA), USA) gave a regulatory perspective
on the S/N approach. Titer assessment, where a serum sample
is serially diluted until it reaches assay background is histori­
cally the most widely used approach for quasi-quantitation of
ADA responses to biotherapeutics and vaccines. This
approach is typically undertaken on serum study samples
that have been screened and confirmed positive using
a tiered immunogenicity testing approach. In recent years, S/
N-based approaches have been proposed and used by specific
pharmaceutical industry stakeholders as a titer surrogate for
quasi-quantitation of ADA responses. This approach is often
used in the absence of subsequent sample titering, which can
be challenging from a regulatory authority and/or health care
practitioner perspective. This presentation provided
a regulatory perspective from the Office of Biotechnology
Products, in the Center for Drugs Evaluation and Research
(CDER) at FDA, whose purview includes assessing the suit­
ability of immunogenicity assays used to test clinical study
samples for biologics and drugs regulated by FDA. From
CDER’s perspective, S/N is a novel alternative to titer assess­
ment, and as such CDER is still gathering experience to estab­
lish its scientific merits and build regulatory confidence in the
approach. Sponsors may use S/N as a titer alternative provided
they suitably justify their choice for use of S/N for ADA quasi-
quantitation in various sections of the regulatory dossier,
including eCTD 5.3.14 Reports of Bioanalytical and

Analytical Methods for Human Studies, 2.7.1 Summary of
Bioanalytical Methods and 5.3.5.3 Integrated Summary of
Immunogenicity. Sponsors are recommended to discuss the
choice of approach with CDER during immunogenicity pro­
gram development through a Type C or Type D meeting
request focused on immunogenicity issues. As part of the
meeting package, Sponsors are recommended to submit sup­
porting ADA assay development data, including: 1) S/N and
titer development data, with a correlation between S/N and
titer-based measurements using an appropriate anti-drug pro­
duct antibody control; 2) early clinical study data correlating
the effect of ADA on PK using both S/N and titer-based
approaches, which can be generated using clinical samples
from a PK study to demonstrate suitability of S/N approach
as an alternative to titer-based approach; and 3) the S/N
criteria established for assigning study samples as treatment-
boosted ADA positive, should these be required.
Until the use of S/N gains wider acceptance from regulatory
agencies, sponsors are still advised to proceed with the cur­
rently recommended tiered immunogenicity assay approach,
including development of a titering assay for ADA quantita­
tion. Given the wide acceptability and conceptual understand­
ing from health care practitioners for the use of antibody titers
to communicate ADA magnitude, an outstanding issue that
awaits clarity is the reporting of S/N values in product labels as
per recommendations in the 2022 immunogenicity draft label­
ing guidance.3
An alternative to neutralizing antibodies (NAbs) assays
Dr Karin Nana Weldingh (Novo Nordisk, Denmark) pre­
sented two case studies supporting the assessment of the neu­
tralizing effect of ADA utilizing an integrated data approach
instead of an in vitro NAb assay. It is important to assess the
neutralizing potential of ADAs, especially for drugs with an
endogenous counterpart. However, an in vitro NAb assay may
not provide the most clinically relevant results because, for
example, binding antibodies without in vitro neutralizing
activity may impair drug activity by enhancing clearance.
Furthermore, in vitro NAbs may not have any clinical effect
since the translation into in vivo situation can be complex. In
vitro NAb assays often include pre-treatment steps altering the
sample matrix and the drug concentration used in the assay is
very different from the therapeutic levels. As an alternative,
companies may suggest evaluating the neutralizing effect of the
ADA by using an integrated data approach in which the
impact of ADAs on exposure, efficacy, and safety is correlated
to ADA levels and persistence of ADA. The first case study
demonstrated that binding antibodies to one part of the drug
molecule resulted in a lack of drug effect by increasing clear­
ance, which was not detected in the in vitro Nab assay.
Correlating ADA levels with PK, PD and clinical effect pro­
vided a more relevant assessment of the clinical effect of the
ADA. The second case was an example of assessing the neu­
tralizing effect of ADAs in the pivotal Phase 3 trials using well-
established read-outs for PK and PD. This approach was
accepted by health authorities providing that the ADA samples
were banked, assay validations were considered appropriate,
and the clinical data demonstrated that PK and PD assays were
MABS 3
sufficiently sensitive to assess a neutralizing effect. In both
examples, PK and PD were considered superior to in vitro
NAb in determining clinically relevant ADA.
Understanding impact
Dr Gregor Lotz (Roche, Germany) introduced novel advanced
ADA characterization assays to understand the impact on
exposure and activity of T-cell engager bispecific drugs
(TCBs). The understanding of ADA-specific drug domain
binding can be critical for the development of TCBs. ADA-
TCB complexes are different in formation and activity when
ADAs are directed either to the target-binding drug domain
site or CD3 activation drug domain-binding site. Therefore,
rapid characterization of ADA domain specificity binding is of
value to understand change in exposure and drug activity.
Biolayer interferometry is a useful tool for rapid characteriza­
tion of ADA domain specificity assessment. This methodology
allows sample analyses of multiple cycles with all domains and
drug in one run. Additionally, the determination of ADA
binding kinetics (kon/koff rates) is possible. Another orthogo­
nal ADA characterization assay was developed to determine
specifically ADA-TCB complexes that increase nonspecific
CD3-mediated activation of T cells and are only induced via
ADAs directed to the target domain of the drug. The detection
of these specific complexes along with ADA domain binding
specificity data allows a better understanding of nonspecific
ADA complex-induced activity and helps the clinical team to
potentially de-risk and adapt mitigation strategies during early
clinical development.
Update from the EIP assay strategy group
Dr. Joanna Grudzinska-Goebel (Bayer AG, Germany) pre­
sented the update on the ongoing activities of the EIP
Immunogenicity Risk Assessment Working Group on behalf
of the Working group. The group aims to provide
a harmonized framework for the immunogenicity risk assess­
ment (IRA) of biotherapeutics throughout product develop­
ment based on the experiences from different pharmaceutical
and biotech companies and examples available from literature.
Further, the team intends to provide practical guidance on the
focus of the IRAs performed at different stages in development
and its translation into immunogenicity risk mitigations and
testing strategies facilitating an aligned assessment of compar­
able biotherapeutic molecules throughout life-cycle
management.
Non-clinical immunogenicity risk assessment
Update from the non-clinical immunogenicity risk
assessment (NCIRA) working group
Dr Sebastian Spindeldreher (Integrated Biologix, Basel) pro­
vided an update on behalf of the NCIRA working group. The
group is currently focusing on two main areas. The first area is
centered around standardizing and harmonizing in silico and
in vitro methods to evaluate immunogenicity risk. The work­
ing group continues their work following the NCIRA
4 S. TOURDOT ET AL.
publication on possible harmonization of antigenicity assays,
in order to address regulatory requests and expectations
regarding the validation of assays assessing innate and adaptive
immune responses.4 Their aim is to provide updated overviews
on existing tools, outlines of potential future applications,
generic protocols where possible, and recommendations for
appropriate and inappropriate use, maximizing comparability
between labs and increasing confidence by establishing good
practices. The assays assessed include innate immune response
modulating impurities (IIRMI) assays, dendritic cell matura­
tion assays, in silico tools, MHC-associated peptide proteomics
(MAPPs), T cell and B cell assays.
In the second area of focus, the working group is working
on generating an immunogenicity database. Currently, data on
the same therapeutic is stored in different repositories, includ­
ing public repositories such as publications, labels, clinical trial
databases and registries, as well as in private repositories. The
EIP is part of the Immunogenicity Database Collaborative
(IDC), which is a global, cross-industry consortium compris­
ing pharmaceutical and biotechnology companies and acade­
mia. Its purpose is to create an open-access, uniform, and
curated database that encompasses clinical and pre-clinical
immunogenicity information for protein-based therapeutics.
It is a grassroots initiative led by volunteer members and
contributors, and holds no formal association to any single
organization or industry working group. The mission of IDC is
to establish a shared and easily accessible database cataloging
descriptors and relevant data associated with the immunogeni­
city of biotherapeutics, to make clinical and pre-clinical immu­
nogenicity data easily accessible to support the development of
safe and effective biotherapeutics.
Ideally, immunogenicity mitigation will start as early as the
drug design stage. A variety of tools are available to assess the
risk of immunogenicity of candidates and select the one with
the most favorable immunogenicity profile. When it comes to
protein therapeutics, these tools, which consist of in silico
prediction algorithms and in vitro assays assessing the
sequence or impurities-related risk factors, are widely used
across industry. The main sequence de-risking strategy here
is the identification and removal of CD4 T-cell epitopes. This
strategy is validated by post-hoc risk assessment analyses of
molecules with immunogenicity in the clinic, and the study of
brolucizumab was discussed in this context.5 The next step to
further increase the confidence in using in vitro assays as
screening tools for drug design is harmonization of protocols
across laboratories. Concerted efforts by the American
Association of Pharmaceutical Scientists (AAPS)
Immunogenicity Risk Assessment and Mitigation group and
the EIP Non-clinical Risk Assessment working group were
presented and deeper dives took place at the “Bring your
own problems” round table discussions.
Critical parameters of in vitro assays
Dr Sofie Denies (SD Analytics, Belgium) discussed statistical
principles for determining an adequate number of donors to
assess immunogenicity preclinically. It was explained that
HLA-coverage cannot be the decisive factor in determining
sample size because one donor with a certain HLA cannot
predict how all individuals carrying the same allele will react.
Instead, sample size calculations should be based on statistical
principles that link sample size to variability in the outcome of
interest. For assays that report percentage of positive donors,
this is the binomial distribution. During the discussion, it was
emphasized that the binomial distribution can only describe
the distribution of outcomes in experimental samples relative
to the outcome of the same assay, with the same analysis
method, in the entire population. It cannot model the outcome
of one assay relative to the outcome of another assay/clinical
manifestation of immunogenicity. This is also not relevant for
sample size calculations, as increasing sample size can only
produce estimates that are closer to the true value of the
specific assay and cannot compensate for a bias of one assay
versus other assays or clinical immunogenicity. Examples of
how the binomial distribution can be used to determine sam­
ple sizes that allow specific research questions to be answered
were discussed, and a free online tool to perform these calcula­
tions has been made available on shiny.sd-analytics.org/sam
ple size.
A regulatory perspective on critical parameters of in vitro
assays
Dr Daniela Verthelyi (FDA, USA) described key concepts in
the use of in silico and in vitro tools instead of clinical trials to
assess the residual uncertainty linked to potential differences
in product- and process-related impurities of follow-on pep­
tides, proteins, and oligonucleotides. Dr Verthelyi described
the various platforms available and the critical assay attributes
that need to be considered when they are used to inform an
immunogenicity assessment exercise. Dr. Verthelyi stressed
the need to establish that the assays used are fit for purpose
and yield sensitive, specific, reproducible, and traceable data.
Further, drawing from the experience with generic synthetic
peptide submission, it described the type of information that
should be included in regulatory submissions to support their
use and the most frequent deficiencies observed. To illustrate
the type of data expected, several case studies of exercises to
assess innate immune response modulating impurities in ther­
apeutic proteins and peptides were discussed, underscoring the
importance of assessing multiple parameters linked to inflam­
mation, activation of antigen-presenting cells, and cellular
stress to understand whether there are differences in impuri­
ties that could render a product produced using a different
manufacturing process more immunogenic. Lastly, the need to
homogenize testing platforms, generate common controls, and
develop improved statistical tools and models to integrate the
orthogonal data provided by the studies was mentioned.
Standardization of in vitro assays
Laurent Malherbe (Eli Lilly, USA) reported on a joint initiative
led by the Health and Environmental Sciences Institute
Immuno-Safety Technical Committee and the AAPS to
develop a novel reference antibody panel for preclinical immu­
nogenicity risk assessment. One of the challenges for the devel­
opment and comparison of preclinical in vitro
immunogenicity risk assays is the lack of availability of

standard positive and negative control therapeutic proteins for
use in assay qualification and as benchmarks for comparison of
relative immunogenicity. The talk summarized the proposed
reference panel of three lyophilized mAbs known to elicit
different rates of ADA response in clinic and the ongoing
pilot project to validate these standard reagents in T cell assays
performed in 11 different laboratories. The outcome of this
pilot work would be a collaborative industry manuscript com­
paring the performance of the reference panel antibodies
across different platforms and different laboratories. The long-
term goal is to extend the validation to more laboratories
including EIP members and characterize the performance of
the reference panel antibodies using in vitro assays measuring
innate immune cell activation.
Round table discussion
Over 20 attendees gathered to discuss the ins and outs of non-
clinical immunogenicity risk assessment and the discussion
reflected current hot topics in the field. Key questions,
answers, and comments are summarized below.
Genetic background as a risk factor: Some companies are
now HLA genotyping subjects in Phase 3 clinical trials to have
enough power to conduct correlation analyses between HLA
and immunogenicity to the drug. For such analyses in the
hemophilia field, the registry of 8,000 hemophilic patients,
which exists in the UK, can be a data source. Beyond HLA
genotyping, correlation analyses with genotyping of Fcgamma
receptors and complement could inform the safety risk.
Gene therapy: Host immune responses should be taken in
consideration as they may influence the long-term immuno­
genicity to the transgene. As well, codon modifications have
been found to alter immunogenicity of the encoded protein
and could therefore be considered an additional risk factor to
assess. Interestingly, regarding the risks linked to the vector,
MHC class I epitopes derived from AAV capsids have been
studied by MAPPs and the analysis revealed peptides of unu­
sual lengths. The reason for longer peptides has not been
elucidated yet.
Regulatory requirements: Currently, the use of non-clinical
immunogenicity risk assessment tools is only requested by US
FDA for generic peptides drug per ANDA guidance.6 For this,
in vitro assays need to be fit-for-purpose validated. However,
the field is moving toward increased harmonization, qualifica­
tion, and validation of the most commonly used in vitro tools
at large (see above), and this might lead to the extension of
regulatory requirement to other biologics.
Application of the assays: Beyond risk assessment of the
amino-acid sequence and structure/format of the biologics,
the field is evolving toward application of the cell-based
in vitro assays to address product critical quality attributes.
This could be applied during the design phase (detection of
post-translational modifications) or later during clinical devel­
opment to assess, for instance, the impact of production pro­
cess changes on the risk of immunogenicity.
The one-million-dollar question: If you could run only 2
in vitro assays, which ones would they be? The unanimous
answer was that the non-clinical risk assessment strategy
should be tailored to each program, hence those 2 assays will
MABS 5
vary upon the context, the drug, and the stage of product
development. Also the question of whether it even makes
sense to run any assays should be posed.
The most common question: Are the assays predictive of
clinical immunogenicity? The answer is a resounding no. The
suite of assays is designed to assess some of the risks linked to
the product, such as the presence of CD4 T cell epitopes,
aggregates, and capacity to activate APCs. However, the overall
immunogenicity of a program is based on other factors that are
taken into consideration, including numerous treatment- and
patient-related factors. The entirety of these factors will be
reviewed and recorded in the Integrated Summary of
Immunogenicity (ISI) to conclude on an overall risk of
a product to induce unwanted immune responses, including
ADA, and their consequences on safety and efficacy.
Immunogenicity of novel modalities
Gene therapy
Cell and gene therapy products (CGTs), named Advanced
Medicinal Therapy Products in the European Union, are
a diverse group of biotherapeutics designed to modify or
manipulate the expression of a gene to treat conditions for
which there are limited or no effective treatment available.
Dr. Laura I. Salazar-Fontana’s (LAIZ Reg Science,
Switzerland) presentation delineated how the risk assessment
principles applied to monitor, evaluate, and mitigate the
immunogenicity of therapeutic proteins and peptides can
also be applied to this new product modality. The main take-
home message was that humoral adaptive immune responses
evidenced by the presence of treatment-emergent ADA are not
sufficient to address the immunogenicity of CGTs. Whilst
preexisting antibodies directed to the viral vector or against
components of lipid nanoparticles (e.g., PEG) used in the
delivery of the genetic material should be evaluated and ana­
lyzed in the context of hypersensitivity reactions (e.g., comple­
ment system and Fcgamma receptor activation), innate (e.g.,
TLR system) and adaptive cellular responses to the two com­
ponents of GT products, the viral vector and the transgene
(e.g., CD8 ± specific T cells), have also proved to be critical
read-outs for the long-term safety and efficacy of this type of
biotherapeutics.
Gene editing
Dr Zuben Sauna (CBER, FDA, USA) discussed understanding
and navigating immune responses to Cas proteins used in gene
editing. The immunogenicity assessment of novel modalities,
e.g., Cas proteins used in gene editing, are likely to present
unique challenges. These proteins are of microbial origin, and
thus are in the high immunogenicity risk category per FDA.7
Moreover, preexisting antibodies and memory T cell responses
have been demonstrated to Cas9 proteins derived from
Staphylococcus aureus or Streptococcus pyogenes. ,8–10 Some
but not all lessons learned from immunogenicity assessments
of therapeutic proteins can be directly applied to Cas proteins.
For instance, many applications of Cas proteins will require
that these proteins are delivered as mRNA and the protein will
6 S. TOURDOT ET AL.
be expressed intracellularly by target cells. A workflow to
identify Cas9-derived peptides presented on single HLA
Class I variants expressed on a monoallelic cell lines was
presented. These data can be compared to those obtained in
previously published data wherein purified Cas9 protein was
incubated with dendritic cells and the HLA Class II associated
Cas9 peptides were isolated.11 These studies on presentation of
Cas-derived peptides on HLA Class I and II can be followed up
with T cell proliferation assays to identify T cell epitopes on
Cas proteins. Taken together, extant and emerging predictive
in vitro assays can estimate the probability of an immune
response to Cas proteins used therapeutically. However,
immunogenicity risk also requires that the clinical conse­
quences of these immune responses (if they occur) are
assessed. Studies to determine the in vivo consequences of
preexisting immunity to Cas remain a critical unmet need.
mRNA-LNP
Dr Arno Kromminga (BioNTech, Germany) discussed the
immunogenicity risk assessment of mRNA-LNP products.
The administration of mRNA is a novel technology with
a proven record in infectious diseases and promising results
in oncology and other disease entities.12 The concept of in vivo
generation of the active compound avoids multiple manufac­
turing- and formulation-related issues. In addition, it allows
the combination of multiple compounds and domains without
challenging structural issues. Furthermore, it enables the use of
a plethora of different mRNA constructs, including unmodi­
fied mRNA (uRNA), backbone optimized mRNA (modRNA),
self-amplifying mRNA (saRNA), and trans-amplifying mRNA
(taRNA).13 The latter enables the expression of multiple inde­
pendent active compounds. Besides infectious diseases,
mRNA-based medicinal products can be used for numerous
oncology indications, including melanoma, prostate cancer,
and non-small cell lung cancer. Even more exciting is indivi­
dualized Neoantigen-Specific Immunotherapy (iNEST) in
which individual mRNA from cancer patients are used for
the personalized stimulation of immune responses.
Regardless of the disease indications, all constructs inherently
need to be encapsulated for administration and several options
are available: lipid nanoparticles (LNP) are commonly used,
but also lipoplexes and polyplexes are under investigation.
LNP contain a substantial amount of PEG (1–5%). As it is
known that the prevalence of preexisting antibodies is as high
as 90% post-CoV2 vaccination there is an intrinsic risk of
boosting an immune response against PEG using LNP as
vehicles for mRNA. However, to date there are no data avail­
able consistently showing that anti-PEG antibodies have an
impact on PK parameters or on clinical consequences of the
medical treatment. If anti-PEG antibodies needed to be mon­
itored, the bioanalytical strategies may not follow the normal
3-tiered approach. Namely the determination of a screening
assay cut point is hampered by the high prevalence of preex­
isting anti-PEG antibodies. In addition, it is more efficient to
run a screening and confirmatory assay in parallel. Also, it is
conceivable to avoid the use of an assay cut point at all and
using titer increases instead. Finally, the determination of anti-
PEG antibodies might be helpful to interpret the potential
clinical consequences of anti-PEG antibodies. Beyond the
immune response against the vehicle, the determination of
the antibodies against the mRNA translatable product is chal­
lenging because the antigens need to be generated (and is not
readily available as a GMP product) for use in an immuno­
genicity assay. Consequently, not only the assay positive con­
trols are surrogate compounds, but also the assay antigens to
be used for the antibody detection against mRNA-translatable
product. Theoretically, immune response against the surrogate
antigens and the mRNA translatable compound generated
in vivo may differ, and hence need to be controlled.
Immunogenicity risk assessment regulatory
considerations
Dr Lydia Michaut (Novartis, Switzerland) shared some overall
feedback received by EIP member companies from health
authorities on their dossiers. The biological consequences of
the development of an immune response to biological thera­
peutic agents have been shown to decrease treatment efficacy
and threaten patients’ safety. Over time, assessment of the
immunogenicity risk carried by any new biological entity has
become an essential part of submission dossiers. Regulatory
authorities have issued guidelines to help pharmaceutical and
biotechnology companies conduct such assessment in
a comprehensive and integrated manner. Guidelines undergo
revision cycles as the field progresses and novel modalities and
vectors emerge. For sponsors, the challenge lies in translating
the guidelines into internal procedures and presenting the
collected immunogenicity data in a clear, concise, and inte­
grated manner to facilitate regulatory review.
Conclusion and outlook
The 14th European Immunogenicity Platform Open Symposium
connected experts and newcomers across academia, industry,
and regulatory agencies to share experience and knowledge to
overcome immunogenicity challenges. The diversification of
biologics and emergence of novel modalities, including cell
and gene therapy products, brings along additional complexity
in the immunogenicity risk assessment and testing during drug
development. The topics discussed included approaches to sim­
plify the immunogenicity testing via use of singlicate analysis, or
S/N data as substitute for titer evaluations; case examples of
integrated analysis of ADA with PK and PD as alternative for
Nab evaluation and characterization of ADA to multi-domain
therapeutics to evaluate clinical impact; and dedicated sessions
on non-clinical immunogenicity assays and new modalities,
including cell and gene therapies and mRNA-LNPs. Via the
annual open symposium and the working groups, EIP aspires
to provide a forum where innovative approaches, learnings, and
regulatory expectations for immunogenicity monitoring, clinical
relevance evaluation and immunogenicity risk evaluation can be
shared and further discussed within the community.
Acknowledgments
The EIP would like to extend their warm thanks to all speakers and
attendees of the 14th Open Symposium for their active participation and
 MABS 7

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the company. Refer to Listed Drugs of rDNA Origin
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Integrated Biologix GmbH ase Cas9 in the USA population. Mol Ther Methods Clin Dev.
ZS reports no conflict of interest 2018;10:105–12. doi:10.1016/j.omtm.2018.06.006.
VS is a full-time employee of UCB Biopharma SRL and might hold 9. Charlesworth CT, Deshpande PS, Dever DP, Camarena J,
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Funding
human population. Nat Med. 2019;25(2):242–8. doi:10.1038/
The author(s) reported there is no funding associated with the work s41591-018-0204-6.
featured in this article. 11. Simhadri VL, Hopkins L, McGill JR, Duke BR, Mukherjee S,
Zhang K, Sauna ZE. Cas9-derived peptides presented by MHC
class II that elicit proliferation of CD4(+) T-cells. Nat Commun.
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