ABSTRACT
Biologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, 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 assessment, 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 longevity of this annual meeting highlights how the development 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, multispecific, conjugated antibodies, novel scaffolds, alongside cell and gene therapy products, including those for gene editing has brought additional complexity to immunogenicity 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, including assay development and analysis for clinical immunogenicity 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 immunogenicity community. The topics and discussions are presented in this report.
Immunogenicity monitoring and clinical relevance
Given the potential consequences of the development of an unwanted immune response, confident monitoring of immunogenicity 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.Citation1 that included data from several immunogenicity assays and clinical studies from different pharmaceutical 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 simulations 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 preexisting 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 titration. 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 validation by assessing the factors impacting S/N and titer, such as assay range, precision, drug/target interference, and low affinity/avidity.
Mr. Daniel Baltrukonis (Pfizer, USA) provided an industry perspective by building upon the publication by Manning et al.Citation1 ADA data across 17 clinical programs spanning 5 formats/modalities (standard monoclonal antibody (mAb)), fusion proteins, bispecifics, antibody-drug conjugates, and gene therapies (anti-adeno associated virus) and both electroluminescence (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 correlation 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 limitations 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 observations 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.Citation2
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 historically 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 suitability 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 assessment, and as such CDER is still gathering experience to establish 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 program 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 supporting 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 product 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 currently recommended tiered immunogenicity assay approach, including development of a titering assay for ADA quantitation. Given the wide acceptability and conceptual understanding 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 labeling guidance.Citation3
An alternative to neutralizing antibodies (NAbs) assays
Dr Karin Nana Weldingh (Novo Nordisk, Denmark) presented two case studies supporting the assessment of the neutralizing 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 clearance, which was not detected in the in vitro Nab assay. Correlating ADA levels with PK, PD and clinical effect provided a more relevant assessment of the clinical effect of the ADA. The second case was an example of assessing the neutralizing 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 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 characterization 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 orthogonal 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) presented 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 assessment (IRA) of biotherapeutics throughout product development 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 comparable 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) provided 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 working group continues their work following the NCIRA 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.Citation4 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 maturation 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, including 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 comprising pharmaceutical and biotechnology companies and academia. 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 immunogenicity of biotherapeutics, to make clinical and pre-clinical immunogenicity 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.Citation5 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 sample sizes that allow specific research questions to be answered were discussed, and a free online tool to perform these calculations has been made available on shiny.sd-analytics.org/sample 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 peptides, 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 therapeutic proteins and peptides were discussed, underscoring the importance of assessing multiple parameters linked to inflammation, activation of antigen-presenting cells, and cellular stress to understand whether there are differences in impurities 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 immunogenicity risk assessment. One of the challenges for the development 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 comparing 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 immunogenicity 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 unusual 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.Citation6 For this, in vitro assays need to be fit-for-purpose validated. However, the field is moving toward increased harmonization, qualification, 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 development to assess, for instance, the impact of production process 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 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 analyzed in the context of hypersensitivity reactions (e.g., complement system and Fcgamma receptor activation), innate (e.g., TLR system) and adaptive cellular responses to the two components 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.Citation7 Moreover, preexisting antibodies and memory T cell responses have been demonstrated to Cas9 proteins derived from Staphylococcus aureus or Streptococcus pyogenes. ,Citation8–10 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 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.Citation11 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 consequences 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.Citation12 The concept of in vivo generation of the active compound avoids multiple manufacturing- 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 unmodified mRNA (uRNA), backbone optimized mRNA (modRNA), self-amplifying mRNA (saRNA), and trans-amplifying mRNA (taRNA).Citation13 The latter enables the expression of multiple independent 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 individualized 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 available 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 monitored, 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 preexisting 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 challenging because the antigens need to be generated (and is not readily available as a GMP product) for use in an immunogenicity assay. Consequently, not only the assay positive controls 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 therapeutic 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 integrated 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 simplify 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 valuable contributions to the discussions, as well as to Barbara Vercruyssen for her invaluable assistance with planning the conference. Presentations can be found on the EIP website.
Disclosure statement
ST and DB are full-time employees of Pfizer Inc. and might hold shares of the company.
SD reports no conflict of interest
VD reports no conflict of interest
JGG. is a full-time employee of Bayer and holds shares of the company
AK is a full-time employee of BioNTech and might hold shares of the company
GPL is a full-time employee of Roche Diagnostics GmbH and might hold shares of the company.
Laurent M is a full-time employee and stockholder of Eli Lilly and Company
Lydia M is a full-time employee of Novartis and might hold shares of the company.
KNW is an employee and a shareholder in Novo Nordisk A/S
JAP reports no conflict of interest
LISF reports no conflict of interest
SS is a full-time employee, partner, and managing director of Integrated Biologix GmbH
ZS reports no conflict of interest
VS is a full-time employee of UCB Biopharma SRL and might hold shares of the company.
DV reports no conflict of interest
DK is a full-time employee of Sanofi Aventis Deutschland GmbH and might hold shares of the company
Additional information
Funding
References
- Starcevic Manning M, Hassanein M, Partridge MA, Jawa V, Mora J, Ryman J, Barker B, Braithwaite C, Carleton K, Hay L. et al. Comparison of titer and signal to noise (S/N) for determination of anti-drug antibody magnitude using clinical data from an industry consortium. AAPS J. 2022;24(4):81. doi:10.1208/s12248-022-00728-8.
- McCush F, Wang E, Yunis C, Schwartz P, Baltrukonis D. Anti-drug Antibody Magnitude and Clinical Relevance Using Signal to Noise (S/N): Bococizumab Case Study. AAPS J. 2023;25(5):85. doi:10.1208/s12248-023-00846-x.
- FDA. 2022. Immunogenicity information in human prescription therapeutic protein and select drug product labeling — content and format. Guidance for Industry - draft guidance.
- Ducret A, Ackaert C, Bessa J, Bunce C, Hickling T, Jawa V, Kroenke MA, Lamberth K, Manin A, Penny HL. et al. Assay format diversity in pre-clinical immunogenicity risk assessment: toward a possible harmonization of antigenicity assays. Mabs-austin. 2022;14(1):1993522. doi:10.1080/19420862.2021.1993522.
- Karle AC, Wrobel MB, Koepke S, Gutknecht M, Gottlieb S, Christen B, Rubic-Schneider T, Pruimboom-Brees I, Leber XC, Scharenberg M. et al. Anti-brolucizumab immune response as one prerequisite for rare retinal vasculitis/retinal vascular occlusion adverse events. Sci Transl Med. 2023;15(681):eabq5241. doi:10.1126/scitranslmed.abq5241.
- FDA. 2021. ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin
- FDA. 2014. Immunogenicity Assessment for Therapeutic Protein Products - Guidance for Industry.
- Simhadri VL, McGill J, McMahon S, Wang J, Jiang H, Sauna ZE. Prevalence of pre-existing antibodies to CRISPR-Associated nuclease Cas9 in the USA population. Mol Ther Methods Clin Dev. 2018;10:105–7. doi:10.1016/j.omtm.2018.06.006.
- Charlesworth CT, Deshpande PS, Dever DP, Camarena J, Lemgart VT, Cromer MK, Vakulskas CA, Collingwood MA, Zhang L, Bode NM. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 2019;25(2):249–54. doi:10.1038/s41591-018-0326-x.
- Wagner DL, Amini L, Wendering DJ, Burkhardt LM, Akyuz L, Reinke P, Volk HD, Schmueck-Henneresse M. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat Med. 2019;25(2):242–8. doi:10.1038/s41591-018-0204-6.
- 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. 2021;12(1):5090. doi:10.1038/s41467-021-25414-9.
- Liu J, Liu Y, Xia H, Zou J, Weaver SC, Swanson KA, Cai H, Cutler M, Cooper D, Muik A. et al. BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants. Nature. 2021;596(7871):273–5. doi:10.1038/s41586-021-03693-y.
- Sahin U, Kariko K, Tureci O. mRNA-based therapeutics — developing a new class of drugs. Nat Rev Drug Discov. 2014;13(10):759–80. doi:10.1038/nrd4278.