Replacing Animal Models with Human-Relevant New Approach Methodologies (NAMs) for Immunogenicity in Clinic

Global Regulatory Agency Perspective on NAMs as a Human-Relevant, Risk-Based Framework for Immunogenicity

Recent FDA guidance and parallel global regulatory perspectives signal a clear shift toward New Approach Methodologies (NAMs) as credible and increasingly preferred tools for immunogenicity risk assessment—particularly for biologics and emerging molecular modalities where animal models have historically lacked predictive value.

The FDA’s 2025 Roadmap to Reducing Animal Testing in Preclinical Safety Studies and its 2026 draft guidance General Considerations for the Use of NAMs in Drug Development together establish a policy and scientific foundation for replacing or supplementing animal studies with human-relevant methods.

A central driver for this shift is recognition that animals are often poor predictors of drug product-based human immune responses.  Moreover, >90% of drugs fail in humans despite promising animal data, suggesting target and relevant drug disposition related differences. For immunogenicity specifically—where species specific differences in sequence, structure, and immune cells can drive anti-drug antibody (ADA) responses—this limitation is particularly acute.

The FDA explicitly identifies NAMs as capable of evaluating immunogenicity, alongside toxicity and pharmacodynamics, using human‑relevant systems, including:

• in silico models (e.g., T cell epitope prediction)
• in vitro human cell assays (e.g., whole blood, PBMC, specialized APCs)
• advanced platforms (e.g., organoids, organ‑on‑chip)

The 2026 guidance reframes regulatory expectations around “fit‑for‑purpose” validation, meaning NAMs can be accepted if they are appropriate to answer a specific immunogenicity risk question (e.g., T cell activation, cytokine release), rather than requiring full replacement of animal testing. Key points include:

• Context of use: clearly defining the immunogenicity risk decision supported
• Human biological relevance: demonstrating mechanistic alignment with human immune responses
• Technical characterization and reproducibility
• Weight‑of‑evidence integration alongside other data sources 

Importantly, the FDA now encourages inclusion of NAM data in IND submissions, and in some cases allows these approaches to partially or fully replace animal studies—particularly for monoclonal antibodies and other biologics with limited animal translatability.

This stance aligns with the broader consensus of global agencies, who have long recognized the limitations/low predictivity of animal models for human immunogenicity and have encouraged a multidisciplinary, integrated assessment, combining nonclinical, analytical, and clinical data.

Across regions (FDA, EMA, MHRA, ICH, PMDA), immunogenicity assessment is converging on a few consistent principles:

1. Clinical immunogenicity (ADA/nAb incidence and impact) remains the gold standard but must be predicted and mitigated earlier.
2. Nonclinical animal data have limited predictive value for human immunogenicity risk, especially for biologics.
3. Mechanistic understanding (e.g., T cell epitopes, target engagement and immune modulation, aggregation, impurities) of immunogenicity risk is central to risk assessment.
4. Integrated, weight‑of‑evidence frameworks are expected.

Taken together, regulatory guidance points toward a tiered immunogenicity risk strategy increasingly centered on NAMs for biologics, peptides, emerging modalities, generics/biosimilars, and critical quality attributes (CQAs), including: early-stage risk identification using computational tools, mechanistic in vitro confirmation, analytical characterization, translation of predicted immune activation to clinical outcomes, and supporting data integration to inform clinical plans. It is suggested that immunogenicity should not be viewed solely as a late-stage clinical observation, but rather as a mechanistically informed, human-relevant risk continuum that can be interrogated throughout development using NAMs to characterize specific components of immune activation before clinical exposure and to inform optimized bioanalytical and clinical strategies.  Further, it is encouraged for these NAMs to be incorporated transparently into regulatory submissions.

EpiVax Capabilities: Mapped to Regulatory NAM Expectations

EpiVax’s platform capabilities are highly aligned with this regulatory direction, offering a comprehensive NAM toolkit spanning in silico and in vitro immunogenicity risk assessment.

T cell Epitope-Driven Risk Prediction

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 immunoinformatics 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, moving beyond assessment to actionable mitigation
• Long retrospective and prospective validation history of superior predictive performance supported by peer-reviewed publications, highlighting 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

Regulatory Fit: Early risk identification and mitigation, human relevance

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 for 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 give first insights into regulatory narratives, and predictive modeling complements in vitro assays rather than replacing them. 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.

In Vitro Human Cell-Based Assays

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 human-based 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

Regulatory Fit: Experimental confirmation with orthogonal methods, human relevance

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 an immunogenicity risk assessment while aligning with regulatory expectations. 

The in vitro human assays developed by EpiVax, such as the human PBMC assay for peptides and impurities, have gone through rigorous reviews and feedback from regulators and align well with the principles described in the Organization for Economic Co-operation and Development (OECD) framework for NAM acceptance. Specifically, they incorporate defined biological systems, standardized operating procedures, donor characterization, assay controls, reproducibility assessments, and mechanistic endpoints linked to adverse outcome pathways involving T-cell-mediated immune responses. These human PBMC assays exemplify the type of scientifically robust, human-centric methodology envisioned by both OECD and FDA initiatives to advance predictive toxicology while reducing reliance on animal testing. 

Regulatory Support

EpiVax scientists have extensive experience in compiling multi-phase NAM analyses into high impact regulatory submission content for Immunogenicity Risk Assessment (IRA) sections of IND submissions and Integrated Summary of Immunogenicity (ISI) sections of BLA submissions. With insights into the required information and format regulators expect, EpiVax can help to interpret the evolving NAM guidances related to immunogenicity assessment into an integrated, weight-of-evidence framework for risk characterization in these documents. EpiVax supports these activities via our TEA Consulting and IRA/ISI writing services.

Bottom Line

Regulators are transitioning immunogenicity assessment toward a NAM‑enabled, human‑centric paradigm. Animal data are no longer considered sufficient or reliable predictors of immune response risk, and agencies are explicitly encouraging mechanistic, epitope‑driven, and cell‑based approaches within a fit‑for‑purpose, weight‑of‑evidence framework.

Within this landscape, EpiVax’s capabilities map directly to regulatory expectations by providing:

• In silico epitope prediction for sequence-based risk identification
• In vitro human cell-based assays for mechanistic validation
• Integrated assessment and reporting expertise for translation of individual analysis methods into a comprehensive risk assessment to be utilized for clinical planning and preparation of regulatory-ready reports

As a result, EpiVax’s NAM toolkit can serve as a central pillar of modern immunogenicity risk assessment strategies, supporting IND‑ and BLA-enabling packages that align with both the FDA’s roadmap and evolving global regulatory standards.