Immunogenicity Risk Assessment Outputs to Drive a Streamlined, Risk-Based Preclinical and Clinical Strategy

Overview: A Strong Immunogenicity Risk Assessment Strategy Starts Early and Carries through Development

Unexpected immunogenicity can derail even the most promising therapeutics in the clinic. Whether it’s a high titer ADA signal, a neutralizing response, hypersensitivity or safety related immune reaction or an unexplained PK shift, surprises in mid- to late-stage clinical development often trace back to missed opportunities in preclinical development. Fortunately, these surprises are increasingly preventable.

Drug developers are now expected to consider immunogenicity and accompanying risks not as a single study endpoint, but continuously from discovery to commercialization, to assess and address risk factors early and as they emerge throughout development. Global regulatory agency guidances emphasize providing a comprehensive immunogenicity risk assessment leveraging human-relevant new approach methodologies (NAMs), such as in silico models and in vitro assays, to inform downstream strategies at IND submissions.  

The incorporation of immunogenicity insights throughout the process allows developers to identify potential issues, focus resources on critical risks, and design studies that are both efficient and scientifically defensible. As such, outputs from the in silico and in vitro risk assessments are informing how developers should approach IND-enabling studies and later stage development phases, resulting in significant return on investment through risk-based streamlining of bioanalysis in the clinic.

Instead of being reactive during clinical studies when unexpected immune responses appear, teams can now anticipate them, refine their candidates for such intrinsic and extrinsic liabilities, and enter the clinic with a clear expectation of risk. Such robust risk assessment can influence a streamlined bioanalytical and clinical immunogenicity strategy, where the focus can shift from “catch all”, indiscriminate testing to a leaner, data-driven method that emphasizes robust testing when risk is meaningful and a more refined approach when it’s not.

EpiVax scientists have key insights into how immunogenicity risk assessment can translate into substantial preclinical and clinical stage efficiencies and savings, and we are here to help from start to finish with best-in-class in silico, in vitro, consulting, and regulatory support expertise.

Read on for more context on why immunogenicity risk assessment data is critical to successful therapeutic development and regulatory submission and for highlights of what EpiVax brings to the table as a preferred end-to-end immunogenicity risk assessment and clinical development partner.

Where Immunogenicity Risk Assessment Data Matters in the Regulatory Submission Process

Executing immunogenicity risk assessment steps “early and often” is both practical and increasingly aligned with regulatory expectations, especially in light of the recently published NAMs guidelines. From a development standpoint, understanding potential immune liabilities before conducting involved IND-enabling regulated studies and clinical testing may avoid development of involved bioanalytical assays or untimely program failures. From a regulatory perspective, agencies increasingly expect sponsors to provide a thorough Immunogenicity Risk Assessment (IRA) leveraging NAMs as part of their IND submission and an Integrated Summary of Immunogenicity (ISI) as part of their BLA, with an aim to prioritize the identification, mitigation, and monitoring of patient safety risks.

Multiple data outputs from preclinical risk assessments can be combined to establish the foundation of an IRA:

• In silico analyses implemented to predict candidate and impurity sequence adaptive immunogenicity risk.
  • EpiVax’s industry-leading ISPRI toolkit can identify T cell epitopes, evaluate the level of T cell epitope self vs non self tolerance threshold, flag and mitigate intrinsic sequence risk, benchmark against clinically relevant molecules, and inform downstream in vitro assay design.
• In vitro assays implemented as orthogonal methods of assessment to evaluate immune responses to candidate sequences and product CQAs in various platforms.
  • EpiVax’s suite of modular, scalable in vitro assays can assess innate and adaptive immune response features like cellular activation, cellular proliferation, and cytokine secretion in human whole blood, PBMC, and specialized APC-based platforms.

The IRA serves as a summary of assessments of the baseline immunogenicity risk profile and can be updated throughout clinical development as new data on observed immune responses and clinical impact emerge, ultimately supporting preparation of the ISI. As immunogenicity risk is a key determinant of whether a therapeutic can safely enter human trials, the IRA is critical for justifying the implementation of a more tailored bioanalytical and clinical strategy where possible.

The ISI functions as a living document, bringing together the evolving risk profile driven by changes in drug product lots and associated critical quality attributes, changes in formulations, dosing regimens and route of administration, consequent bioanalytical strategy, and clinical data generated throughout development to support a comprehensive immunogenicity assessment and inform risk management and regulatory decision-making. The development of an ISI is essential as the definitive characterization of immunogenicity across the entire development program can determine labeling and post-market requirement parameters.

EpiVax scientists have extensive experience in the preparation of this high impact regulatory submission content, with insights into the required information and format regulators expect. EpiVax supports these activities via our TEA consulting and IRA/ISI writing services.

Integrating In Silico Analysis with ISPRI into the IRA

EpiVax’s proprietary ISPRI ™ in silico toolkit provides teams with a detailed view of amino acid sequence-based adaptive immunogenicity risk. Using best-in-class, internally and externally validated algorithms, ISPRI is utilized to evaluate candidate sequences and impurity sequences for T cell epitope density and tolerance potential, generating a comprehensive immunogenicity risk profile for as few as one sequence to as many as thousands of sequences in seconds.

Key features of the ISPRI toolkit include:

• Integrated, proprietary immunoinformatic engines that enable mechanistic T cell epitope-driven risk prediction and optimization. Key AI/ML driven algorithms include:
  • EpiMatrix → predicts HLA Class I and II T cell epitopes (core driver of ADA risk)
  • ClustiMer → identifies T cell epitope “hotspots” (high-risk regions)
  • JanusMatrix → evaluates T cell epitope human homology/cross-reactivity (distinguishes immunogenic vs tolerated or tolerogenic epitopes)
  • OptiMatrix → enables hotspot deimmunization/humanization (sequence redesign to de-risk liabilities)
• High-throughput to in-depth level screening – real-time analysis with quantitative scoring and benchmarking:
  • The toolkit generates normalized scores to enable: 1) rapid ranking and triaging of large candidate sets and 2) detailed analysis and benchmarking of candidates against known immunogenic and non-immunogenic therapeutics (context is key)
    • Enables portfolio-level decision-making
    • Integrates immunogenicity as a front-end selection filter, not a late-stage risk
    • Enables tailored analyses aligned to development stage and modality
• Sequence optimization and humanization capabilities move beyond assessment to actionable mitigation
• Long retrospective and prospective validation history of superior predictive performance supported by peer-reviewed publications – transparency and real-world use
  • Depth of validation and regulatory credibility is unmatched
  • Modeling of immune tolerance reduces false positive/negatives and improves clinical relevance
  • Results are interpretable and mechanistically explainable – critical for regulatory defensibility

EpiVax offers several service options that enable self-service analysis or EpiVax-led assessments fit for different stages of development. For example, the ISPRI Downselect™ report supports high-throughput screening of many candidates during lead selection, filtering out those with high predicted risk. For more advanced programs, the ISPRI Evaluate™ report provides a comprehensive analysis suitable for IND submission, often paired with in vitro validation.

These predictive results inform specific next steps integrating with experimental and regulatory workflows:

• Assay design, ensuring that downstream in vitro and clinical bioanalytical assays are appropriately scaled and fit to assess previously identified risk factors.
• Clinical monitoring strategy, helping teams anticipate when and where immune responses are most likely to appear.

In silico analyses provide first insights into regulatory narratives, and predictive modeling complements in vitro assays rather than replacing them. By using in silico insights to design in vitro experiments, developers can prioritize the most informative assays and reduce redundancy, creating a more time- and cost-efficient plan.

Integrating In Vitro Assays with EpiVax into the IRA

At the core of EpiVax’s in vitro offerings are three complementary human-relevant NAM assay platforms, whole blood, PBMC, and specialized APC systems (macrophages/DCs), each providing distinct insight into immune activation. Whole blood captures integrated innate responses in a clinically relevant setting. PBMC assays enable evaluation of both innate and adaptive immunity, including T cell activation and population-level (HLA-dependent) variability. APC-based systems provide mechanistic insight into antigen processing, presentation, and early signaling that drives downstream responses.

Key assays include:

Whole Blood Assay: Evaluates cytokine release following exposure to a test article using multiplexed analysis (Luminex), providing early insight into innate activation and immune overstimulation risk in a clinically relevant system. Commonly applied to assess immunotoxicity (e.g., cytokine storm with T cell engagers/CAR-T) and innate responses to CQAs (e.g., impurities, aggregates, or novel chemistries).
Modalities: Broad compatibility (mAbs, bsAbs, ADCs, cytokines, CAR-T constructs, peptides, nucleic acids, delivery vectors).
Use Cases: Candidate selection, first-in-human (FIH) risk mitigation, impurity assessment, comparability/manufacturing changes, formulation screening.

PBMC Assay: A modular platform assessing innate and/or adaptive immune responses via cytokine profiling (Luminex/FluoroSpot) and cell activation/proliferation (flow cytometry). Design parameters (donors, duration, readouts) can be tailored to development stage and modality. Frequently used alongside in silico tools to assess immunogenicity risk and innate-to-adaptive priming.
Modalities: Non-immunomodulatory proteins (mAbs, bsAbs, enzymes, hormones, CAR-T domains, HCPs), peptides, nucleic acids, mRNA/LNP, viral capsids.
Use Cases: Candidate optimization, FIH risk mitigation, impurity characterization, comparability studies.

DC:PBMC Assay: Uses dendritic cell–PBMC co-culture to assess adaptive immune responses through cell activation/proliferation and cytokine profiling. Particularly valuable for immunogenicity risk of immunomodulatory products; can be simplified to DC activation alone or extended with immunopeptidomics (MAPPs).
Modalities: Immunomodulatory proteins (mAbs, bsAbs) and peptides (especially unnatural/cyclic).
Use Cases: Candidate selection, FIH risk mitigation, comparability studies, formulation/route/dose optimization.

Class II HLA Binding Assay: Cell-free assay measuring peptide binding affinity to recombinant HLA Class II alleles via fluorescence. Used to validate in silico predictions and assess peptides (especially with unnatural amino acids) not suited for computational methods.

PANDA® Screening: A tailored solution for ANDA sponsors developing generic peptides, aligning with FDA requirements to assess immunogenicity risks from APIs and impurities.

• Class II HLA Binding Assay: Assesses peptide–HLA binding affinity
• T Cell Assay: Measures peptide-induced T cell responses (PBMC-based)
• Innate Immune Response Assay: Evaluates innate activation using PBMC

Together, these platforms form a modular assay suite that can be strategically applied across development stages—from early risk characterization to late-stage comparability—to evaluate specific aspects of immunogenicity risk based on modality and program needs. This flexible approach supports targeted risk assessment, reduces unnecessary in vivo/clinical testing, and enables data-driven decisions, especially when integrated with in silico predictions to de-risk development and strengthen the IRA while aligning with regulatory expectations.

Translating Preclinical Insights into the IRA

When preparing an IND, the Immunogenicity Risk Assessment (IRA) section serves to combine predictive and experimental data from preclinical development into a regulatory-ready composition.

A well-supported IRA includes:

• A clear summary of identified risk factors and corresponding mitigation strategies, such as sequence deimmunization, formulation refinement, removal of co-eluted host cell proteins, and specification setting for degradation- and post-translational modification-related critical quality attributes.
• Justification for the proposed bioanalytical assays and clinical monitoring plans.
• A rationale for ongoing risk evaluation throughout later phases.

Regulators expect more than raw data. A strong IRA explains how results shaped downstream choices, not just what testing was performed during clinical stage of development. Pairing in silico and in vitro insights to obtain the relevant outputs shows that immunogenicity risk is being proactively addressed and mitigated rather than reactively managed in later stage trials.

EpiVax’s analyses and assays, which always conclude with a comprehensive report, can help fill IND data gaps. EpiVax experts can compile this information into a specific, industry- and regulatory-aligned IRA format, which can later be updated into an all-encompassing ISI.

Leveraging the IRA to Inform Bioanalysis and Clinical Planning

Immunogenicity risk assessment doesn’t end at IND submission, and it’s not just a regulatory check box. The most successful programs maintain continuous data integration from preclinical to clinical phases and beyond.

Insights gained from an integrated in silico and in vitro preclinical assessment approach should be leveraged as a comprehensive framework to inform efficient bioanalytical and clinical strategies, rather than following a “one-size-fits-all” plan. Together, the risk assessment outputs should generate a weight-of-evidence dataset that can guide bioanalytical (e.g., ADA assay risk tiering, selection of relevant biomarkers, and monitoring plans) and clinical (e.g., first-in-human starting dose, patient monitoring, and risk mitigation plans) factors.

As an example, the IRA can inform an evidence-based tiering approach for ADA assay strategy. Low risk molecules with minimal predicted immunogenicity may warrant a “collect and hold” approach in early clinical phase development, where samples are collected from patients but only assessed if an event may warrant investigation, and a more streamlined ADA assay approach with less frequent monitoring in later clinical phase development. Whereas high risk molecules may require a much more robust ADA monitoring approach with high frequency sampling and high sensitivity assay development required even in earlier stages of clinical development.

With a complete preclinical risk assessment, bioanalytical and clinical strategies can oftentimes be streamlined to significantly reduce timelines and costs without compromising safety.

A Continuous, Informed Approach to Immunogenicity Risk with EpiVax

EpiVax’s predictive tools and in vitro assays make it possible to anticipate and mitigate risk, not just measure it. When integrated into IND creation and bioanalytical design, they provide a data-driven foundation for regulatory confidence and clinical success.

EpiVax scientists frequently collaborate with development, CMC, and bioanalytical teams to ensure these connections are made early. With the right preclinical data in hand, teams can move into the clinic not just faster, but smarter.

Still not convinced? Find out more about why you should choose EpiVax as your end-to-end immunogenicity risk assessment partner.