The number of approved or investigational viral vector gene therapies (GTx) continues to grow. The adeno-associated virus vector (AAV) technology is one of the most applied GTx platforms. Presence of pre-existing anti-AAV immunity is viewed as a potential barrier for successful AAV treatment. Presentation will cover questions related to risk-based approach in assessment of anti-AAV cellular immunogenicity, evidence of correlation between humoral and cellular immune responses, and details related to broadly used analytical methodologies, including assay validation and performance parameters. 

Preexisting antibodies derived from prior exposure to adeno-associated viruses (AAV) may limit successful vector transduction and reduce efficacy of AAV-vectored gene therapies. In this presentation, we explain the rationale for the selection of a Companion Diagnostic (CDx) for an AAV5-based gene therapy encoding for human FVIII intended for treatment of Hemophilia A (valoctocogene roxaparvovec, BMN 270), and present its real-world utility in assessing global prevalence of preexisting immunity to AAV5.

Cell and Gene Therapy drug development has led to an expanded interest in cellular immunogenicity as part of regulated bioanalysis. Flow cytometry and ELISpot are playing a much larger role in the characterization of the patient's immune response to these therapies and leveraging these tools leads to a number of new challenges around sample collection, storage and shipment logistics, and complex multiparameter analyses which will be discussed in this talk.

Novel modalities such a lipid nanoparticle encapsulated mRNA where the in vivo transcribed proteins are expressed on the cell membrane or intracellularly making neutralizing antibody methods impossible to develop. Reliance on surrogate endpoints, such as biomarkers as measures of neutralization can be used. This talk will discuss NAb strategies based on immunogenicity risk and examples of alternative approaches for assessment of neutralizing antibodies based on two case studies.

Immune-receptor specific engineered cytokines that take up the role of endogenous pleotropic cytokines, that are crucial for innate and adaptive immune responses for cancer and inflammation are interesting and at high-risk. While promising, modified cytokines have the potential to be immunogenic and generate NAbs against endogenous counterparts too. More challenges to establish a cell-based assay, and relevant MOA based cell line itself to detect NAbs are limiting factors. To overcome the hurdles, a novel ready-to-use (RTU) functional cell-based assay was developed to test NAbs against cytokine-mutein drugs. A method qualification NAb assay case study will be presented in this talk.

The current testing paradigm for anti-drug antibodies in clinical samples follows a tiered approach where all confirmed ADA binding positive samples should be further characterized with respect to e.g. neutralizing activity. The assays used for detection of binding antibodies are usually very sensitive and not as prone to drug interference as cell based neutralizing antibody assays representing mode of action. Sensitivity and particularly sensitivity in the presence of drug are often in a different range in such NAb assays as compared to binding antibody assays. This presentation will discuss different scenarios with respect to sensitivity of both bAb and NAb assays, titers detected in the bAb assay and number of samples to be analysed. To ensure generation of meaningful results that can be related to the potential clinical impact, we suggest an alternative strategy for selecting relevant clinical samples for in vitro NAb testing in different scenarios.

Immunogenicity assessment for bispecific therapeutic antibodies is challenging, particularly in cell-based assays (CBA) for NAb detection. Multiple factors may interfere in NAb detection such as drug, soluble target, and matrix effects. This presentation will discuss several analytical approaches to mitigate interference including target depletion, NAb extraction, and analytical variability based cut point calculation. The comparison between LBA and CBA platforms for bispecific therapeutics NAb detection will be discussed.

Immunogenicity developed against therapeutic biologics is undesirable and can have serious clinical consequences. To address the concern of negative impact of immunogenicity on treatment effects, therapeutic drug monitoring has been adopted as a mechanism of patient management in some clinical specialties. Advances in leveraging clinical pharmacology data have offered an option for evaluating clinical impacts and contribute the considerations of therapeutic drug monitoring. With available and emerging methodologies in the clinical pharmacology evaluations of immunogenicity impact, forward planning towards harmonization and developing best practice is essential. This presentation will discuss the current challenges and potential future directions based on the review experience in the Office of Clinical Pharmacology at the FDA. 

We reviewed the immunogenicity section on the labels of human therapeutic antibodies approved by FDA. Incidence of anti-drug antibodies (ADA) reported in majority of the labels for human monoclonal antibody drugs is consistent with false positive rates predicted by computer simulations for ADA-negative population of a comparable size. This indicates that false positive rates currently recommended for immunogenicity testing may result in over-reporting of ADA incidence.

Drug-induced immunogenicity is a significant concern affecting safety and efficacy of biologic therapies. With improvements in screening assay sensitivity and drug tolerance, reliable estimation of anti-drug-antibody (ADA) titer is required for understanding clinically relevant ADA levels. In this talk, we investigate how titer variability depends on the cut point factor, propose a standardized cut point factor to increase precision, and demonstrate advantages of a non-linear regression approach to estimate titer.

Clinically relevant ADAs can impact PK, efficacy, and safety of biologics. Therefore, it is essential to evaluate and report clinically relevant immunogenicity information. However, lack of harmonization in collecting, analyzing, and reporting of ADA data limit the assessment of clinical consequences. To address inadequate reporting of immunogenicity, a harmonization strategies of data assessment and reporting is required. This presentation will discuss key challenges and strategies regarding immunogenicity data analysis, highlighting the clinically relevant ADA determination strategies.