Immunogenicity Testing: Definition, Guidelines, and Anti-Drug Antibody Detection

Immunogenicity refers to the ability of a drug and/or its metabolites to induce immune responses or immune-related events against itself or related proteins^[1]. The consequences of such immune reactions can range from the emergence of clinically insignificant anti-drug antibodies (AD As) to severe, life-threatening adverse events. Immunogenicity testing is integral to drug development, spanning the entire lifecycle from discovery and nonclinical studies to clinical trials and post-marketing surveillance. ADAs serve as the cornerstone for immunogenicity evaluation. Modern biologic therapies, including monoclonal antibodies (mAbs), fusion proteins, recombinant proteins, gene therapies, novel vaccines (e.g., mRNA vaccines), and cell therapies, represent emerging molecular modalities with inherent immunogenicity risks. This article delineates the principles of immunogenicity, its mechanisms, detection strategies aligned with FDA/EMA/NMPA immunogenicity guidance, and analytical challenges in ADA detection.

What is an anti-drug antibody?

Anti-drug antibodies (ADAs) are immune system-produced antibodies that specifically bind to therapeutic drugs, potentially altering their pharmacokinetics, efficacy, or safety. ADAs can be categorized based on their functions:

• Binding antibodies: Attach to the drug without directly blocking its activity.

• Neutralizing antibodies (NAbs): Inhibit the drug’s biological function by blocking target interaction.

ADAs are particularly concerning for biologic therapies due to their potential to trigger hypersensitivity, reduce drug efficacy, or cross-react with endogenous proteins.

Therapeutic protein drugs can elicit both innate and adaptive immune responses. Innate immunity is a nonspecific, non-memory defense mechanism that constitutes the first line of host defense. However, adaptive immunity—particularly humoral immunity mediated by B-cell-derived ADAs—is the primary focus of immunogenicity testing. As illustrated in Figure 1, ADA generation typically involves T-cell-dependent or T-cell-independent pathways. These pathways often interact, as immune complexes or aggregates can concurrently activate both mechanisms.

Figure 1. Mechanism of anti-drug antibody formation^[2].

Factors influencing immunogenicity and its impact on drug development

Early therapeutic antibodies (e.g., murine-derived antibodies) exhibited a high immunogenicity risk due to their foreign origin. Advances in bioengineering—such as chimeric (mouse-human), humanized, and fully human antibodies—have mitigated but not eliminated immunogenicity risks^[3]. Factors influencing immunogenicity are categorized as patient-related (immune status, genetic predisposition, pre-existing antibodies, dosing regimen, route of administration) and drug-related (product origin, structural integrity, aggregation, glycosylation, pegylation, impurity profiles, formulation, storage). The United States Pharmacopeia (USP) General Chapter <1106> stratifies these factors into low-, medium-, and high-risk categories (Table 1).

Table 1. Risk factors for immunogenicity ^[4].

Anti-drug antibodies can profoundly affect drug exposure, pharmacokinetics, efficacy, and toxicity. For instance, ADA-drug complexes may accelerate drug clearance, reduce half-life, or neutralize therapeutic activity. Severe cases may precipitate hypersensitivity reactions, autoimmune disorders, or immune complex deposition-related pathologies^[5].

Immunogenicity testing: regulatory guidelines and strategies

For the detection of ADA, regulatory guidelines from the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the National Medical Products Administration (NMPA, China) advocate a tiered ADA testing strategy: screening, confirmatory, and characterization (Figure 2). Screening identifies potential positives, confirmatory assays validate specificity (e.g., competitive inhibition), and characterization evaluates titer, neutralizing activity, isotype/subtype, and epitope binding. Method validation parameters (e.g., screening cut-point, confirmatory cut-point, sensitivity, precision, selectivity, drug tolerance, specificity, robustness, reproducibility, and stability) align across guidelines, though NMPA/FDA immunogenicity guidance stipulates more granular requirements compared to EMA (Table 2).

Figure 2. Tiered approach for ADA testing ^[1].

Table 2. Validation parameters across regulatory guidelines ^[6-8].

Note: +: with relevant parameters -: not mentioned

Notably, NMPA mandates in vitro cytokine release assays (e.g., IL-2, IL-6, TNF-α) for drugs targeting immune cells or Fcγ receptors, whereas FDA immunogenicity guidance/EMA provides limited guidance on such assays^[1][6-8].

Platform and assay format selection for immunogenicity testing

Regulators require high sensitivity, specificity, robustness, and drug tolerance for anti-drug antibody detection. Common analytical platforms include enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence (ECL), radioimmunoprecipitation assay (RIPA), and surface plasmon resonance (SPR). Table 3 compares the advantages and limitations of each platform.

Table 3. Analytical platform comparison for ADA detection

In addition, the FDA reported in 2018 on the submission methods used over the previous five years. Among these, ADA testing predominantly utilized ECL and ELISA platforms, while neutralizing antibody testing (direct and bridging methods, see Figure 3) mainly relied on cell-based assays and competitive ligand-binding assays.

Figure 3. ELISA direct method

Figure 4. ECL bridging method

Case study: 

An ECL bridging assay was validated for detecting ADAs in rats administered trastuzumab. The method demonstrated a sensitivity of 0.382 ng/mL and drug tolerance of 200 μg/mL (positive control: 500 ng/mL). Among 24 samples (6 rats, 8 timepoints), 8 screened positive, 4 confirmed positive, and titers ranged from 10,200 to 68,000 (Table 4).

Table 4. Summary of test results

Note: / stands for the experiment was not performed.

Overcoming challenges in immunogenicity testing

Positive control antibodies are essential for assessing the performance of detection methods, directly influencing their sensitivity and drug tolerance levels. Choosing an appropriate positive control antibody is crucial for developing and validating ADA detection methodologies. Typically, polyclonal antibodies derived from immunized animals are used for this purpose.

Therapeutic protein drugs present significant challenges for immunogenicity assessment due to their high dosing and long half-lives, which can lead to elevated drug concentrations in circulation. This can interfere with ADA testing, as excess drug may bind to ADA, resulting in false-negative results. To enhance drug tolerance and minimize the risk of false negatives caused by free drug presence, various methods are employed in ADA analysis.

ADA can exist in two forms within a sample: free ADA and ADA-drug complexes. To reduce interference during ADA detection, sample pre-treatment is necessary. Factors such as the characteristics of each drug, detection objectives, risk assessment, sensitivity targets, operational complexity, and feasibility should be considered when selecting analytical methods. In Table 5, we compare the effects of different treatment methods on drug tolerance.

Table 5. Drug tolerance by the pre-treatment method

Additionally, for monoclonal antibody drugs, interference from soluble targets presents a significant challenge. Soluble dimers or multimers of drug targets can create bridges between two labeled drug molecules, leading to false-positive results for ADA. Conversely, if an antibody can only bind to one of the targets, steric hindrance may result in false-negative outcomes. A common approach to address this issue is to remove the soluble targets from the matrix. This is typically achieved using target inhibitors, usually corresponding anti-target antibodies, which bind to and eliminate the interference from the targets, thereby improving target tolerance ^[10].

A final word

Immunogenicity remains a pivotal challenge in the development of biologic therapies. Cross-disciplinary collaboration—spanning bioanalysis, immunology, pharmacology, and regulatory science—is essential for robust risk assessment. WuXi AppTec DMPK provides comprehensive preclinical bioanalytical support for immunogenicity, including ADA method development/validation for monoclonal/bispecific antibodies, antibody-drug conjugates (ADCs), oligonucleotides, and other biologics, ensuring compliance with FDA//EMA/NMPA requirements for investigational new drug (IND) submissions.

Authors: Miaomiao Song, Jie Liu, Xue Zhang, Maotian Zhou, Lili Xing

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