Development of a Cell-based Neutralizing Antibody Assay for Zinpentraxin Alfa: Challenges and Mitigation Strategies.

Therapeutic protein drugs can potentially induce immune responses in patients and result in the production of anti-drug antibodies (ADAs), including a subset of ADAs called neutralizing antibodies (NAbs) that might cause loss of efficacy by inhibiting clinical activities of the drug. Herein, we describe the unique challenges encountered during the development of a fit-for-purpose cell-based NAb assay for a new protein modality, zinpentraxin alfa, including our strategies for assay design to overcome various matrix interferences and improve assay drug tolerance. We demonstrated that a typical biotin-drug extraction with acid dissociation (BEAD) approach alone was not sufficient to eliminate matrix interferences in this assay. Instead, the combination of the BEAD and ZebaTM spin size exclusion plate (SEP) was required to achieve the desirable assay performance. We also demonstrated that appropriate acidic buffers were critical in sample pretreatment to improve assay drug tolerance, which not only dissociated the drug/NAb immune complex but also effectively and irreversibly denatured the free drug. The final assay performed well with confirmed assay robustness and suitability for the clinical applications.

Development of Gene Therapy Vectors: Remaining Challenges.

Almost 20 years after the tragic death of a young patient due to an experimental gene therapy trial to treat Ornithine Transcarboxylase deficiency, the FDA approved its first landmark gene therapy drug i.e. Luxturna® to treat inherited blindness, and dozens of gene therapy studies are underway. Whether it is replacing the mutant copies of the gene with the wild type one or editing the mutant one in or ex-vivo to elicit the production of functional proteins, numerous viral and non-viral vectors for delivering the gene payload are being evaluated. While, non-viral vectors avoid or mitigate limiting factors such as immunogenicity and the presence of neutralizing antibodies (NAbs), viral vectors such as recombinant adeno-associated viruses (AAVs) have shown early success as a delivery vehicle, because of the overall safety, target specificity, and long-term stability profile. Nonetheless, multiple challenges during the AAV product development and approval process are still looming. AAV serotypes are continuously being engineered which requires multiple cell-based assays to not only assess the neutralizing antibodies (NAb) seroprevalence but also to develop the in-vitro bio potency assays. Hence, we focus on some critical aspects of the AAVs that determine the path forward for pre-clinical and clinical product development.

Detection of Biologically Relevant Low-Titer Neutralizing Antibodies Against Adeno-Associated Virus Require Sensitive In Vitro Assays.

Patients with preexisting anti-adeno-associated virus serotype 8 (AAV8) neutralizing antibodies (NAbs) are currently excluded from AAV8 gene therapy trials. Therefore, the assessment of biologically relevant AAV8-NAb titers is critical for product development in gene therapy. However, standardized assays have not been routinely used to determine anti-AAV8-NAb titers, contributing to a wide range of reported anti-AAV8 prevalence rates. Using a clinical in vitro NAb assay in a separate study, a higher than expected anti-AAV8-NAb prevalence of about 50% was found in international cohorts. This comparative study has a translational character, confirming the biological relevance of anti-AAV8-antibody titers measured by this assay. The significance of low-titer anti-AAV8 NAbs is shown, along with the relevance of the in vitro assay cutoff (1:5) compared with other assays. Importantly, internally standardized reagents and purified AAV8 constructs containing 90% full capsids were used to reduce the effect of empty capsids. It was found that even very low anti-AAV8-NAb titers (<1:5) could efficiently hinder transduction in vivo, demonstrating the importance of sensitive NAb assays for clinical applications. The in vitro NAb assay was found to be more sensitive than an in vivo NAb assay and thus more suitable for patient screening. Additionally, the study showed that anti-AAV8-NAb titers <1:5 were very rare, further supporting the in vitro assay. However, assays using a lower cutoff may still be useful to explain potential variances in transgene expression. These findings support the relevance of the higher than expected prevalence of anti-AAV8 NAbs, highlighting the need for strategies to circumvent preexisting anti-AAV8 NAbs.

Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.

Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs.

A signaling-enhanced chimeric receptor to activate the ICOS pathway in T cells.

Activation of the inducible costimulator (ICOS) signaling pathway in T cells is difficult to assess with bioassays, because most T cell lines do not constitutively express ICOS. Additionally, engagement of ICOS by its natural ligand B7 related protein 1 (B7RP1) is insufficient to elicit ICOS signaling, but requires simultaneous costimulation of the T cell receptor (TCR) to be effective. Here we describe a genetically engineered human T cell line that expresses a chimeric receptor (ICOS-CD3) consisting of full-length human ICOS fused at its C-terminal end to the cytoplasmic domain of human CD3 zeta. When engaged by B7RP1, ICOS-CD3 initiated signaling independently of TCR costimulation and induced substantially more IL-2 secretion in Jurkat T cells compared to wildtype ICOS. We demonstrate that this signaling-enhanced chimeric receptor can be used in simple and sensitive bioassays to detect bioactive B7RP1, anti-B7RP1 drugs, and the presence of corresponding neutralizing anti-drug antibodies.