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.

Introducing dendritic cell antibody internalization as an immunogenicity risk assessment tool.

Aim: Immunogenicity risk assessment assays are powerful tools that assess the relative immunogenicity of potential biotherapeutics. We detail here the development of a novel assay that measures the degree of antibody internalization by antigen-presenting cells as a predictor of immunogenicity. Results & methodology: The assay uses the fluorescence signal from the antibody bound to the outside of the cell as well as inside the cell to determine internalization. To calculate the amount of internalized antibody, the fluorescent signal from the outside was subtracted from the fluorescent signal from the inside, which is referred to as the internalization index. Conclusion: This assay format demonstrated that antibody-based biotherapeutics with higher clinical immunogenicity internalized to a higher degree than therapeutic antibodies with lower clinical immunogenicity.

An integrated approach for characterizing immunogenic responses toward a bispecific antibody.

Bispecific antibodies (bsAbs) recognize and bind two different targets or two epitopes of the same antigen, making them an attractive diagnostic and treatment modality. Compared to the production of conventional bivalent monospecific antibodies, bsAbs require greater engineering and manufacturing. Therefore, bsAbs are more likely to differ from endogenous immunoglobulins and contain new epitopes that can increase immunogenic risk. Anti-A/B is a bsAb designed using a 'knobs-into-holes' (KIH) format. Anti-A/B exhibited an unexpectedly high immunogenicity in both preclinical and clinical studies, resulting in early termination of clinical development. Here, we used an integrated approach that combined in silico analysis, in vitro assays, and an in vivo study in non-human primates to characterize anti-A/B immunogenicity. Our findings indicated that the immunogenicity is associated with epitopes in the anti-B arm and not with mutations engineered through the KIH process. Our results showed the value of this integrated approach for performing immunogenicity risk assessment during clinical candidate selection to effectively mitigate risks during bsAb development.

Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells.

Biotherapeutics, which are biologic medications that are natural or bioengineered products of living cells, have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop antigen-specific anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Here, we describe an in vitro method for assessing the immunogenic risk of a biotherapeutic. We found a correlation between clinical immunogenicity and the frequency with which a biotherapeutic stimulated an increase in CD134, CD137, or both cell surface markers on CD4+ T cells. Using high-throughput flow cytometry, we examined the effects of 14 biotherapeutics with diverse rates of clinical immunogenicity on peripheral blood mononuclear cells from 120 donors with diverse human leukocyte antigen class II-encoding alleles. Biotherapeutics with high rates of ADA development in the clinic had higher proportions of CD4+ T cells positive for CD134 or CD137 than biotherapeutics with low clinical immunogenicity. This method provides a rapid and simple preclinical test of the immunogenic potential of a new candidate biotherapeutic or biosimilar. Implementation of this approach during biotherapeutic research and development enables rapid elimination of candidates that are likely to cause ADA-related adverse events and detrimental consequences.