Target interference is a well-recognized challenge in immunogenicity testing for biotherapeutics, particularly monoclonal antibody (mAb) therapeutics, when soluble targets are present at sufficiently high concentrations as dimers, or when a substantial portion of anti-drug antibodies (ADAs) target epitopes overlapping the drug–target interface. The standard practice to assess target interference involves spiking the soluble target and mitigating its effects using one of several reagents—typically competitive anti-target antibodies, soluble ligands, or decoy receptors. While effective, these reagents are often costly and introduce additional complexity as critical assay components. In this presentation, we present case studies and propose a new strategy.

Immunogenicity assessment remains challenging for complex antibody-based modalities. This presentation discusses ADA troubleshooting across ADCs, T cell engagers, and multi-domain molecules, highlighting assay interference, baseline reactivity, and data interpretation challenges. Practical case examples illustrate modality-specific strategies, integrated assay designs, and cross-functional approaches to support risk-based, fit-for-purpose immunogenicity assessment in drug development. 

Recent advancements in xRNA therapies (ASO, siRNA, mRNA) are expanding therapeutic opportunities across multiple diseases. This expansion increases modality complexity and manufacturing challenges. Individual components and combinations may activate innate or adaptive immunity, with potential safety impact. We will present preclinical assessments to identify immunogenicity risks and evaluate potential immune responses to xRNA during drug development and provide a framework to design xRNA with reduced clinical immunogenicity and improved safety.

Immunogenicity assessment for complex drug modalities, such as oligonucleotides and multidomain therapeutic protein, presents unique bioanalytical challenges. Here we present case studies highlighting challenges such as the use of signal-to-noise and domain mapping to characterize ADA responses. These findings can inform considerations for the development and validation of ADA assays to support complex modalities.

While Fc-attenuation is the industry standard for preventing off-target T cell activation by T cell engager (TCE), we demonstrate that immune complexes formed with anti-drug antibodies (ADAs) can override this protection to elicit potent T cell triggering. These findings reveal a critical interplay between immunogenicity and Fc-biology, providing essential insights for refining TCE immunogenicity risk assessments and bioanalytical strategies.

Heterogeneity in regulatory submissions highlights the need for continued harmonization in evaluating immunogenicity and assessing clinical impact. Clinical pharmacology data have emerged as a sensitive measure for evaluating potential impacts on efficacy. The FDA's Office of Clinical Pharmacology is facilitating harmonization across data submission, analysis, and reporting. Recent reviews show substantial increases in labeling communication of immunogenicity risk and clinical impact communication for newly approved products. This presentation will share ongoing efforts and future collaboration opportunities.

This study compares the immunogenicity of three CRISPR proteins—SaCas9, AsCas12a, and CasΦ—as safer alternatives for clinical gene editing. In 43 healthy North American donors, pre-existing antibody and memory T cell responses were measured using ELISA and IFN-γ ELISpot assays. MHC Class I-presented Cas-derived peptides were identified through monoallelic cell lines and MAPPs proteomics, mapping T cell epitopes across diverse HLA types to characterize immunogenicity risks.

Drug tolerance in immunogenicity assays requires a mechanism-informed, modality-specific approach, as circulating drug and target-mediated interference can compromise ADA detection. Optimized dissociation strategies, including acid dissociation and high ionic strength dissociation (HISDA), improve drug tolerance, with combined approaches enhancing performance in high-drug or high-affinity settings. Case examples across multiple modalities highlight the importance of tailoring strategies to exposure and interference to enable reliable ADA assessment.

Neutralizing antibody (NAb) assays for biotherapeutics are often limited by poor drug tolerance, often driving the need for more complex sample treatment strategies such as biotin-drug extraction and acid dissociation (BEAD). This presentation describes a case study in which critical sample treatment and assay conditions were identified through screening DOE and refined using optimization DOE to balance sensitivity, precision, and drug tolerance. The validated assay achieved sub-100 ng/mL sensitivity and =100 µg/mL drug tolerance, meeting study expectations.

Biotherapeutics have significantly evolved since the approval of insulin by the FDA in 1982. Multi-domain biotherapeutics, including fusion proteins, bi-specifics, tri-specifics, anti-drug antibodies, oligo conjugates and others are revolutionizing treatment of patients, particularly in oncology and auto-immune diseases, delivering on the promise of lower toxicities and/or synergistic effects. However, their complexity brings challenges in the bioanalytical space; for immunogenicity assays, there may be a need to assess which portion/domain of the biotherapeutic is eliciting the immune response. As the bioanalytical industry continues to focus on applying immunogenicity risk-based strategies to support programs in drug development, it is important to remind ourselves of historical practices and consensus and determine if there is a need to provide additional clarity or if practices need to evolve. This presentation will cover past recommendations and future perspectives.

For CAR T cell therapies, immunogenicity assessments often focus on ATA detection without fully leveraging B-cell epitope mapping and clinical outcome characterization to establish relevance. This work introduces a novel, risk-based framework integrating preclinical B-cell epitope mapping, longitudinal ATA characterization, and clinical data mining, demonstrating how immune response incidence, epitope hotspots, and patient immune status can be contextualized to distinguish clinically meaningful responses from modality- and disease-related background immunogenicity.

Historically, health authorities have required highly sensitive assays (100 ng/mL) to detect ADA, even when samples contain drug at trough levels. However, for most protein therapeutics, clinical experience has shown that the important question is whether sufficient ADA is present to alter clinical exposure, safety or efficacy. This talk reviews regulatory guidance, explains ADA-to-drug stoichiometry, and proposes an exposure-based, flexible sensitivity target tied to trough concentrations of recently approved mAbs.

A strategy for justifying clinically relevant specifications will be shared where the potential impact of critical quality attributes (CQAs) of biotherapeutics are looked at holistically. Multiple orthogonal approaches can be utilized to understand the risk of CQA impact to safety, immunogenicity, and efficacy.