Development of a FRET-Based Assay for Analysis of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immunogenicity Risk Assessment

Wen, Yi; Cahya, Suntara; Zeng, Wei; Lin, Joanne; Wang, Xiaoli; Liu, Ling; Malherbe, Laurent; Siegel, Robert W.; Ferrante, Andrea; Kaliyaperumal, Arunan

doi:10.1208/s12248-020-00444-1

Cited by 17 publications

(10 citation statements)

References 27 publications

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“… 7 , 8 To evaluate mAbs internalization and degradation, DC from 13 individual donors were incubated with mAbs in the presence of an activatable fluorescence-quencher probe consisting of goat-anti-human IgG antigen-binding fragments (Fabs) conjugated with the tetramethylrhodamine (TAMRA) (fluorophore)–QSY7 (quencher) pair. 18 , 19 In this assay, TAMRA fluorescence only appears following DC internalization of the antibody/probe complex and subsequent degradation to separate the dye and the quencher. After 24-hours incubation, TAMRA fluorescence was detected in DCs that had been incubated with mAb1, mAb2, mAb3 and control mAb ( Figure 6a ).…”

Section: Resultsmentioning

confidence: 99%

Post-hocassessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms

Walsh

Lannan

Wen

et al. 2020

mAbs

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Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro . All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.

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Section: Resultsmentioning

confidence: 99%

Post-hocassessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms

Walsh

Lannan

Wen

et al. 2020

mAbs

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“…Dendritic cells, have been shown to produce cytokines and upregulate cell surface markers that are associated with antigen presentation and co-stimulation of T-cells when exposed to antibody aggregates ( 38 ). Activation of cells has also trended with some monoclonal antibodies that have shown high immune responses in vitro and in the clinic ( 39 ). THP-1 cells have served as a cell line surrogate of antigen presenting cells.…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity Risk Assessment of Spontaneously Occurring Therapeutic Monoclonal Antibody Aggregates

et al. 2022

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Aggregates of therapeutic proteins have been associated with increased immunogenicity in pre-clinical models as well as in human patients. Recent studies to understand aggregates and their immunogenicity risks use artificial stress methods to induce high levels of aggregation. These methods may be less biologically relevant in terms of their quantity than those that occur spontaneously during processing and storage. Here we describe the immunogenicity risk due to spontaneously occurring therapeutic antibody aggregates using peripheral blood mononuclear cells (PBMC) and a cell line with a reporter gene for immune activation: THP-1 BLUE NFκB. The spontaneously occurring therapeutic protein aggregates were obtained from process intermediates and final formulated drug substance from stability retains. Spontaneously occurring aggregates elicited innate immune responses for several donors in a PBMC assay with cytokine and chemokine production as a readout for immune activation. Meanwhile, no significant adaptive phase responses to spontaneously occurring aggregate samples were detected. While the THP-1 BLUE NFκB cell line and PBMC assays both responded to high stress induced aggregates, only the PBMC from a limited subset of donors responded to processing-induced aggregates. In this case study, levels of antibody aggregation occurring at process relevant levels are lower than those induced by stirring and may pose lower risk in vivo. Our methodologies can further inform additional immunogenicity risk assessments using a pre-clinical in vitro risk assessment approach utilizing human derived immune cells.

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“…Thus, we propose that the CD134/CD137 assay should be used as one of several assays for assessing immune responses to biotherapeutics. Methods such as in silico T cell epitope prediction, DC internalization, 43 and proteomic analysis of major histocompatibility complex-associated peptides proteomics (MAPPs) 44 remain critical for providing an accurate and complete portrait of the immunogenicity of a biotherapeutic.…”

Section: Discussionmentioning

confidence: 99%

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

Cohen¹,

Myneni²,

Batt³

et al. 2021

mAbs

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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.

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Development of a FRET-Based Assay for Analysis of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immunogenicity Risk Assessment

Cited by 17 publications

References 27 publications

Post-hocassessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms

Post-hocassessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms

Immunogenicity Risk Assessment of Spontaneously Occurring Therapeutic Monoclonal Antibody Aggregates

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

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