Biologics are revolutionizing patient care across all disease areas. Optimizing a molecule's immunogenicity risk profile contributes to the successful development and launch of biologics. Designing biologics to be of high quality requires integration of data from multiple sources. Application of in silico and in vitro immunogenicity assays in the discovery stage sets the pathway for success. Treating immunogenicity alongside other developability characteristics avoids the need for deimmunization strategies. Integrating data at each stage of development enables simulations of clinical outcomes, giving confidence for making the right decisions. If necessary, these simulations also inform the need for additional clinical mitigation steps.

Immunogenicity remains one of the most complex and critical challenges in drug design and development, requiring the strategic integration of complementary platforms to effectively mitigate risk for biologics and peptide therapeutics. In this presentation, I will share case studies demonstrating ProImmune’s comprehensive suite of adaptive immunogenicity assessment capabilities.

ProImmune also brings extensive expertise in ultra-sensitive CFSE-based T cell proliferation assays. The ProMap® T cell proliferation assay is routinely used to precisely identify CD4+ T cell epitopes and is filed with the FDA as a Drug Master File (DMF), facilitating streamlined referencing for complex generic peptide ANDA submissions. In addition, the advanced ProScern® Dendritic cell-T cell proliferation assay enables detailed comparative assessment of whole proteins to evaluate immunogenicity risk. The Mixed Lymphocyte Reaction (MLR) assay further complements this toolkit as a cornerstone method for assessing immune modulation.

Together with ProPresent® MHC-associated Peptide Proteomics (MAPPS), REVEAL® HLA-peptide binding and functional T cell assays, these platforms provide a fully integrated and mechanistic understanding of adaptive immune responses. By combining peptide presentation, HLA binding, and T cell activation data, ProImmune delivers a holistic view of immunogenicity risk.

Attendees will gain practical insights into how this integrated suite of assays can be applied to characterize adaptive immunogenicity, supporting informed decision-making and risk mitigation throughout the development lifecycle.

Genetically engineered Salmonella strains are promising vectors for cancer therapy, but their clinical use is limited by host immune responses. A key immunogenic antigen, the phosphatase PhoN, is metabolically essential for bacterial survival in the vitamin B6-depleted tumour microenvironment and thus cannot be simply deleted. While conventional deimmunisation targets surface residues, we present a strategy of allosteric deimmunisation by mutating non-surface, buried residues within the dominant h2 helix T-cell epitope. We hypothesised that the epitope is allosterically coupled to the distal active site gate, allowing for the simultaneous modulation of immunogenicity and enzymatic function. Using a computational pipeline, we designed deimmunised variants and used molecular dynamics (MD) simulations to predict their functional effects. The simulations revealed that mutations in the h2 helix allosterically controlled the gate's flexibility. The enzymatic activities of the deimmunized variants were correlated with the MD predictions: two of the five designed variants exhibited 2~3-fold increases in catalytic function. This work demonstrates that non-surface epitopes can be rationally engineered to not only ablate immunogenicity but also to allosterically enhance protein function.

In this presentation, I will discuss why proactively managing immunogenicity as a critical product attribute is essential for achieving commercial success in drug development.

Immunogenicity is a critical safety concern in the development of peptide-based therapeutics, with the potential to impact both clinical efficacy and patient safety. For Abbreviated New Drug Application (ANDA) submissions, the growing complexity of peptide drugs necessitate integrated immunogenicity risk assessment to support informed decisions throughout development. In silico methodologies enable early identification of sequence- and structure based risk factors, such as major histocompatibility complex (MHC) binding potential, T cell epitope density, and population level HLA coverage. On the other hand, in vitro assay systems such as antigen presentation and T cell activation assays provide mechanistic evaluation of peptide driven adaptive immune responses, including cytokine release and T-cell activation. While in vitro assays enable functional assessment under controlled conditions, key scientific considerations include fit-for-purpose assay selection, robust study design, thorough assay validation, appropriate donor selection, ensuring biological relevance and addressing challenges associated in translating experimental findings across orthogonal approaches. An integrated framework that combines predictive modelling with functional adaptive immunity assays for immunogenicity risk assessment enables a comprehensive understanding of immune risk and supports scientifically justified risk mitigation strategies. These topics, along with practical case insights, will be presented to demonstrate strategies for streamlining study design, enhancing data interpretation, and effectively integrating immunogenicity findings into regulatory submissions.

Comparative in vitro analytical methods to characterize innate immune response modulating impurities can inform the immunogenicity risk and help streamline the development of generic peptides and biosimilar proteins. This talk will describe a new Multiplexed Immunophenotyping for Innate Activation Assessment (MIIAA) assay that utilizes single-cell spectral flow cytometry to capture innate responses by APCs, like cDC, pDC, and other cell types that are critical in initiating immune and inflammatory responses but are at low frequency in peripheral blood. MIIAA offers a powerful immunophenotyping tool to characterize single-cell responses to drug products and potential immunomodulatory impurities that may find utility in drug pipelines to characterize the impact of therapeutics on specific immune cells and to interrogate immunogenic or immunomodulatory risk in comparisons between reference and follow-on products.

Innate immunogenicity assays using fresh whole blood have become a pivotal approach for assessing the immunogenicity risk of biological drugs due to their clinical relevance. These assays are crucial for regulatory filings, especially for generic peptide drugs, as they help identify risks tied to process- and product-related impurities. Unlike peripheral blood mononuclear cell (PBMC) assays, fresh whole blood assays represent all blood cell types without introducing bias from cell purification methods. To ensure that the data reliably inform immunogenicity risk, thorough assay validation is essential - covering sensitivity, drug tolerance, specificity, precision, robustness, and accuracy.

In this talk, I outline the comprehensive process followed at ProImmune to validate fresh whole blood assays. This includes establishing cytokine panels, generating standard curves, defining assay acceptance criteria (such as upper and lower limits of quantification), and confirming signal recovery and assay variability. I will also cover the selection of Innate Immune Response Modulating Impurity (IIRMI) controls, applied both alone and spiked into the drug product to determine assay sensitivity.

Additionally, I discuss the importance of confirming the viability of diverse antigen-presenting cell phenotypes, analyzed via flow cytometry, in the presence of the drug. This also includes the use of cell-death controls to ensure accuracy. Finally, I highlight critical considerations for study design, such as determining the highest drug product concentration that maintains cell viability, the range of drug batches to test, and the IIRMI spiking strategy.

As a leader in this field, ProImmune’s ProStorm Cytokine Release Assay is on file with the FDA as a Drug Master File (DMF), simplifying the reference process for your Abbreviated New Drug Application (ANDA).

Panellists:
Prof. Yoonjoo Choi, Chonnam National Medical University Medical School, South Korea
Dr. Daniela Verthelyi, USA
Dr. Julia Rozenfeld, Teva Pharmaceutical, Australia
Dr. Balaji MR, Dr. Reddy's Laboratories, India

The FDA's roadmap for advancing New Approach Methodologies (NAMs) identifies unwanted immunogenicity as a priority area, emphasizing scientifically validated alternatives to animal testing. Progress in the last two decades has allowed the development of an integrated framework combining in silico, in vitro, and ex vivo NAMs for comprehensive immunogenicity assessment. The tools include: (i) In silico approaches such as T-cell epitope prediction algorithms, AI/ML structural modeling, and population HLA diversity analysis. (ii) In vitro methodologies that encompass dendritic cell-T cell activation assays, cytokine profiling panels, and mass spectrometry-based tools to identify T-cell epitopes. (iii) Ex vivo platforms that utilize HLA-stratified PBMC and whole blood assays from genetically diverse donor cohorts to predict human immune responses. In addition, emerging tools such as small ankyrin repeat proteins called Ankyrons that bind with high affinity to diverse protein targets can be used to interrogate immune mechanisms.

Here I will provide specific examples of the use of NAMs in addressing immunogenicity issues during drug development. I will also discuss the complexities of evaluating the immunogenicity risk of diverse novel modalities and how platform-specific immunogenicity assessment strategies can be developed. Key unmet needs will also be identified and the need for collaborative efforts between industry, academia, and regulatory agencies to standardize tools and develop reference reagents will be emphasized.

MHC-associated peptide proteomics (MAPPs) identifies presented peptides for immunogenicity assessment, though in silico tools often over-predict compared to experimental results. This study demonstrates that antibody modifications can reveal previously "missed" peptides, aligning MAPPs data with predictions. These results suggest that in silico hits are likely valid findings not discovered partly due to limited standard detection sensitivity.

The identification of novel and complex protein targets in drug discovery increasingly exposes the limitations of conventional antibody generation, particularly for targets that are poorly immunogenic or structurally challenging. These challenges can delay target validation and introduce risk into early-stage development. Ankyrons are engineered recombinant binding proteins (~15 kDa) designed to overcome these constraints, providing high-affinity, target-specific reagents in a rapid and scalable format.

Selected from ProImmune’s highly diverse TeraLibrary™ (~10¹² variants) using ribosome display, Ankyrons enable fully in vitro, high-throughput binder generation without the need for immunising animals. This allows accelerated targeting of difficult or novel antigens, including pathogen-derived proteins and emerging therapeutic targets, supporting faster progression from target identification to functional validation.

Their small size and robust biophysical properties offer advantages in tissue penetration and epitope accessibility. Ankyrons are readily deployable alongside antibodies across standard assay platforms including flow cytometry, immunofluorescence, Western Blot, ELISA immunohistochemistry and more. They can be engineered into multivalent or multispecific formats to support more complex assay designs and therapeutic hypotheses.

For drug developers, Ankyrons provide a flexible and cost-effective solution for de-risking early discovery by enabling precise epitope mapping, target engagement studies, and biomarker assay development. Their applicability across multiple species also supports translational studies and cross-species validation strategies.

In this presentation, we will present case studies illustrating how Ankyrons have been applied to previously intractable targets, accelerating target validation, informing candidate selection, and supporting downstream assay development. Attendees will gain practical insight into integrating Ankyrons into drug discovery workflows to enhance speed, confidence, and decision-making.

For therapeutic proteins, peptides, and oligonucleotides, the measurement of anti-drug antibody responses during clinical trials is currently used to identify potential immunogenicity-related adverse outcomes. However, this approach is not suitable for all products or development programs including those for follow-on products. To address these gaps, immunogenicity risk assessment is pivoting towards the use of new analytical, in silico, in vitro, and ex vivo tools. This talk will focus on the information needed to construct an informative risk assessment using the new analytical approaches, describe the possibilities and limitations of these approaches, and discuss how they are being used in regulatory strategies.

The development of anti-drug antibodies (ADAs) remains a significant hurdle in the clinical translation of therapeutic proteins, potentially impacting safety, pharmacokinetics, and efficacy. To address this, a proactive preclinical strategy is essential to de-risk candidates prior to First-in-Human (FIH) trials. We implemented a systematic, three-tiered immunogenicity assessment pipeline:

1. Tier 1 (In Silico): Computational screening of T-cell epitopes to identify high-risk sequences.
2. Tier 2 (In Vitro/Ex Vivo): PBMC-based assays to evaluate the functional immune response in human-derived cells.
3. Tier 3 (In Vivo): Pharmacokinetic (PK) and ADA profiling in cynomolgus monkeys to observe the immune response in a non-human primate model.

We have evaluated the correlation between these three layers across several lead candidates. Each component provides distinct insights into immunogenic potential, with specific strengths and limitations. The value of the pipeline lies not in a single definitive "yes/no" answer, but in the convergence of data to guide protein engineering and lead selection. Although no current preclinical model offers 100% predictive accuracy for human immunogenicity, this integrated pipeline provides a robust framework for ranking candidates. By identifying and mitigating risks early, we can significantly improve the success rate of clinical translation for next-generation biologics.

Antibody therapeutic development requires early control of immunogenicity risks, as most protein drugs can elicit anti drug antibodies that impact efficacy, safety, and pharmacokinetics. This presentation introduces an integrated immunogenicity risk assessment (IRA) approach combining in silico T cell epitope prediction with in vitro DC:T cell assays and biophysical analyses. Together, these methods provide rapid sequence level risk screening and functional measurement of CD4⁺ T cell activation—via proliferation, stimulation above background, and Response Index (RI)—to sensitively identify potential T cell epitopes in antibody candidates.

A case study in collaboration with ProImmune illustrates how these complementary platforms can be operationalized within preclinical programs to inform lead candidate selection. Integrating in silico scoring, DC:T cell assay data, pharmacodynamic profiles, and biophysical properties supports a holistic, data driven decision making framework. Importantly, such integration also provides financial and strategic value: early risk visibility enables developers to better quantify the overall liability of a molecule, reduce late stage failures, and accelerate go/no go decisions—ultimately improving the efficiency and cost effectiveness of program progression.

This talk summarizes practical strategies, assay principles, and key learnings from applying an integrated immunogenicity assessment approach to real world candidate optimization, highlighting both scientific and economic advantages of early, data rich immunogenicity evaluation.

While George Box famously noted that "all models are wrong, but some are useful," this principle is especially resonant in preclinical immunogenicity assessment. In vitro tools like DC/T-cell and MAPPs assays offer critical mechanistic insights, yet their cost and complexity often challenge project timelines and budgets. Drawing from personal experience, I would like to discusses how to implement these assays by moving away from "checking every box" toward a risk-based, cost-of-goods approach.

Immunogenicity remains one of the major uncertainties in biologic development, and once an antibody enters the clinic, opportunities to mitigate an immunogenicity issue are extremely limited. For this reason, we focus our assessment of predictive immunogenicity to the early stages of discovery. At Teva, we routinely screen large panels of antibody sequences to identify potential T-cell epitope liabilities and engineer them out before selecting a lead. This strategy enables rapid elimination of high-risk sequences and ensures that antibodies with no predicted T-cell epitopes advance. This design-first approach has had a meaningful downstream impact: our clinical-stage antibodies consistently demonstrate low ADA rates (<2–5%) and stable pharmacokinetics, underscoring that thoughtful early engineering can prevent costly surprises later. In summary, predictive immunogenicity assessment and upfront design optimization provide each molecule with a stronger chance of clinical success.

Immunogenicity assessment and mitigation ideally results in products launched with minimal anti-drug antibody incidence and no clinical impact of those ADA. Investing in immunogenicity assessment tools at the right time to support successful discovery, development and launch requires an objective view of the return on investment. Prioritization of spending on assays should be determined by the IRA, clear expectations of what will be good enough, and the likelihood of being able to make a positive decision. For Biotechs looking to partner with established Pharma, demonstrating a complete IRA, informed by assays and demonstrated derisking activity will be viewed favourably. Investors are increasingly aware of the risks of immunogenicity and may require IRA and assays.