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ABSTRACT
Therapeutic antibodies sometimes elicit anti-drug antibodies (ADAs) that can affect efficacy and safety. Engineered antibodies that contain artificial amino acid sequences are potentially highly immunogenic, but this is currently difficult to predict. Therefore, it is important to efficiently assess immunogenicity during the development of complex antibody-based formats. Here, we present an in vitro peripheral blood mononuclear cell-based assay that can be used to assess immunogenicity potential within 3 days. This method involves examining the frequency and function of interleukin (IL)-2-secreting CD4+ T cells induced by therapeutic antibodies. IL-2-secreting CD4+ T cells seem to be functionally relevant to the immunogenic potential due to their proliferative activity and the expression of several cytokines. The rates of the donors responding to low and high immunogenic proteins, mAb1, and keyhole limpet hemocyanin were 1.3% and 93.5%, respectively. Seven antibodies with known rates of immunogenicity (etanercept, emicizumab, abciximab, romosozumab, blosozumab, humanized anti-human A33 antibody, and bococizumab) induced responses in 1.9%, 3.8%, 6.4%, 10.0%, 29.2%, 43.8%, and 89.5% of donors, respectively. These data are comparable with ADA incidences in clinical settings. Our results show that this assay can contribute to the swift assessment and mechanistic understanding of the immunogenicity of therapeutic antibodies.
Abbreviations
| ADAs | = | anti-drug antibodies |
| APCs | = | antigen presenting cells |
| CPD | = | cell proliferation dye |
| DAVID | = | Database for Annotation, Visualization, and Integrated Discovery |
| DEGs | = | differentially expressed genes |
| GO | = | Gene Ontology |
| IL | = | interleukin |
| KLH | = | keyhole limpet hemocyanin |
| PCSK9 | = | proprotein convertase subtilisin/kexin type 9 |
| PBMC | = | peripheral blood mononuclear cell |
| 7AAD | = | 7-amino-actinomycin D |
Acknowledgments
The authors would also like to thank Jacob Davis for his technical editing.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Author contributions
C.K. designed the study; Y.A. and C.K. wrote the manuscript; Y.A., S.M., T.I, S.I., M.Y., A.T., and C.K. executed the cellular assay and analyzed the assay data; Y.A., T.M., H.M., N.Y., and Y.N. executed the RNA-Seq experiment and analyzed the data; Z.S. produced and characterized the antibodies; M.T., S.C., M.M., J.K., H.S., and M.H. supervised the study.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2253570.