The number of oviral vector-based gene therapies (GTx) is quickly growing with two products (Zolgensma and Luxturna) already approved in the US. Majority of GTx therapeutics are adeno-associated virus-based (AAV) vectors. Pre-existing humoral immunity against AAVs presents a significant concern as it is expected to impact treatment safety and/or efficacy. Patients are commonly screened for the anti-AAV antibodies in either neutralizing (NAb) or total binding antibody (TAb) detecting assays. Presentation will review current opinions on value of TAb vs. NAb assays, recommendations for the development and validation of anti-AAV TAb methods, parameters critical for monitoring of assay performance.

Immunogenicity assessment is an integral part of biotherapeutics development. Anti-drug antibody (ADA) measurements inform the host responses against the biotherapeutics treatment, and interpretation of ADA results should be performed within the relevant clinical context. This presentation highlights a case study for a bispecific therapeutic with a focus on bioanalytical strategy considerations and analysis of ADA impact on other clinical endpoints.

Several case studies will be discussed that explore the utilization of predictive data for assessing immunogenicity risk to justify the setting or widening of safe specifications for patients in regulatory filings.

While critical reagents are the basis for immunogenicity assays, few characterization guidelines have been provided by regulatory agencies. In addition to determining quality attributes, reagent characterization can enable assay development by guiding reagent selection and assay design to meet sensitivity, drug tolerance, and other criteria. Here, we will describe several reagent characterization approaches and provide examples of how the resultant data have been used to develop and optimize immunogenicity assays.

Immune responses to biologics can change or reduce their efficacy and may increase adverse events. Anti-drug antibodies are associated with clinical immunogenicity. CD4 T cell epitopes contained within the sequences of biologics are the key drivers of immunogenicity. This presentation will address CD4 T cell assays to assess control biologics to demonstrate how T cell dependent immune responses are important in the overall strategy in immunogenicity risk assessment.

Immune responses to biotherapeutics have the potential to affect pharmacokinetics, pharmacodynamics, safety and efficacy of the product. To ensure product quality and to reduce the risks of unwanted immunogenicity, it is imperative to properly and proactively assess and mitigate immunogenicity risk of biotherapeutics. This talk will focus on in silico and in vitro T cell assays to characterize the immunogenic potential of different biotherapeutic proteins and their correlation to the clinically observed outcome

Immunogenicity risk assessment (IRA) is an iterative process that requires a cross-functional effort to identify potential risk factors for immunogenicity and to build appropriate risk evaluation and mitigation strategies for clinical development. Early in drug discovery, in silico, and in vitro approaches are often used to identify immunogenic sequences and to de-risk lead candidates, wherever possible. As the lead candidate moves through development, IRA is reevaluated, and appropriate mitigation tools and de-risking strategies are implemented. This presentation will focus on our thought processes around IRA and mitigation strategies using a low to moderate risk biotherapeutic as a case study.

Polyclonal antibodies are widely used reagents in biotech and pharmaceutical industries due to their excellent performance characteristics and robust stability, but suffer from limited supply and batch-to-batch variability. Similarly, convalescent or vaccinated patient plasma has recently received much attention but met similar reproducibility challenges in clinical applications. Rapid Novor’s REpAb sequencing has overcome many of these challenges through the sequencing and recombinant expression of mAbs found directly from pAb mixtures.

It is important to understand the immune response elicited from cell and gene therapies. For most protein biotherapeutics, humoral immunogenicity is assessed using standard bridging anti-drug antibody assays. However, there has been a greater interest in characterizing cellular immunogenicity responses that has coincided with development of more advanced modalities, requiring the use of an array of different platforms. In this presentation, bioanalytical assays for understanding cellular immunogenicity will be discussed.

An understanding of the potential immunogenicity and immunotoxicity risk of your therapeutic candidate is a key part of pre-clinical development. While human primary cell assays can provide vital data to help support this assessment, the design and qualification of such human in vitro assays is key to generating high quality, reliable data to support both lead selection and regulatory filings.

Cell-based assays (CBA) for NAb are notoriously challenging: drug and soluble targets can generate false-positive/negative results even at low concentrations. In a CD20xCD3 bispecific CBA, prior anti-CD20 therapy (e.g., rituximab) was also shown to interfere. This was mitigated by addition of blocking mAbs, but each anti-CD20 therapy requires specific blockers. This highlights challenges with target-based NAb assays, both from exogenous therapies and endogenous anti-target antibodies (e.g., infectious disease).

Health agencies do request to extend ADA monitoring for patients that developed an ADA response to the drug until ADAs return to baseline levels. However, there is no common understanding in which cases an extension of ADA follow-up sampling beyond the End of Study (EOS)  is required. In this talk, the European Immunogenicity Platform (EIP) provides recommendations under which circumstances such a follow-up of ADA-positive subjects should be considered.

Immunogenicity risk assessment is an essential step in bringing therapeutic drugs to the market. ProImmune's risk management tools evaluate immunogenic epitopes and the corresponding functional T cell responses that can lead to unwanted immune responses. Case studies will highlight how the integrated platform is used to address key questions in the drug development phase. 

The immunogenicity assay package for Biotherapeutics includes screening, confirmation, titer and neutralizing antibody (Nab) assays. Besides such well-known assays, analysis of the ADA specificity might be beneficial in order to understand an immune response and their potential impact. The presentation gives an overview on established and new methods including case studies for illustration of their challenges and chances.

Over the past 15 years, the assessment of anti-drug antibodies (ADA) to biotherapeutics has predominantly used a tiered testing strategy of screen, confirm, and titer as standard practice. However, simplification of this testing strategy is possible, based on today’s improved methodologies and a better understanding of what ADA tiered data provides. Clinical case studies will be presented that support when and why confirmatory and/or titer assays may not be necessary.