Introducing dendritic cell antibody internalization as an immunogenicity risk assessment tool
Abstract
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.
Papers of special note have been highlighted as: • of interest; •• of considerable interest
References
- 1. . The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. MAbs 2(3), 256–265 (2010).
- 2. . Immunologic responses to therapeutic biologic agents. J. Investig. Allergol. Clin. Immunol. 18(5), 335–342 (2008).
- 3. . Immunotoxicity and immunogenicity of biopharmaceuticals: design concepts and safety assessment. Curr. Drug Saf. 5(4), 293–307 (2010).
- 4. . Immunotoxicity of monoclonal antibodies. MAbs 1(2), 104–111 (2009).
- 5. . Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20, 621–667 (2002).
- 6. . The generation of antibody-secreting plasma cells. Nat. Rev. Immunol. 15(3), 160–171 (2015).
- 7. . The ins and outs of MHC class II-mediated antigen processing and presentation. Nat. Rev. Immunol. 15(4), 203–216 (2015).
- 8. . T-cell dependent immunogenicity of protein therapeutics: preclinical assessment and mitigation. Clin. Immunol. 149(3), 534–555 (2013). • One of the first pivotal studies to use in vitro T-cell activation assay to assess immunogenicity.
- 9. Quantitative analysis of the CD4+ T cell response to therapeutic antibodies in healthy donors using a novel T cell: PBMC assay. PLoS One 12(5), e0178544 (2017).
- 10. Considerations for optimization and validation of an in vitro PBMC derived T cell assay for immunogenicity prediction of biotherapeutics. Clin. Immunol. 137(1), 5–14 (2010).
- 11. . Secukinumab, a novel anti-IL-17A antibody, shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity. MAbs 8(3), 536–550 (2016).
- 12. Comparative profiling of HLA-DR and HLA-DQ associated factor VIII peptides presented by monocyte-derived dendritic cells. Haematologica 103(1), 172–178 (2018).
- 13. . MAPPs for the identification of immunogenic hotspots of biotherapeutics; an overview of the technology and its application to the biopharmaceutical arena. Expert Rev. Proteomics 15(9), 733–748 (2018).
- 14. MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity. MAbs 10(8), 1168–1181 (2018). • Dendritic cell culture method and stimulation used as basis for current assay.
- 15. . HLA-DR-presented peptide repertoires derived from human monocyte-derived dendritic cells pulsed with blood coagulation factor VIII. Mol. Cell. Proteomics 10(6), M110.002246 (2011).
- 16. Development of a FRET-based assay for analysis of mAbs internalization and processing by dendritic cells in preclinical immunogenicity risk assessment. AAPS J. 22(3), 68 (2020). •• Details development of a similar assay that looks at both antibody internalization and processing.
- 17. . Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody. Clin. Exp. Immunol. 183(1), 102–113 (2016).
- 18. Cardiovascular efficacy and safety of bococizumab in high-risk patients. N. Engl. J. Med. 376(16), 1527–1539 (2017).
- 19. Effects of RG7652, a monoclonal antibody against PCSK9, on LDL-C, LDL-C subfractions, and inflammatory biomarkers in patients at high risk of or with established coronary heart disease (from the phase 2 EQUATOR study). Am. J. Cardiol. 119(10), 1576–1583 (2017).
- 20. Phase I study of anticolon cancer humanized antibody A33. Clin. Cancer Res. 9(4), 1338–1346 (2003).
- 21. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities. MAbs 4(3), 326–340 (2012).
- 22. . The function of Fcγ receptors in dendritic cells and macrophages. Nat. Rev. Immunol. 14(2), 94–108 (2014).
- 23. . Actin filaments facilitate two steps of endocytosis. J. Cell Sci. 109(Pt 2), 457–465 (1996). • Technique used to inhibit macropinocytosis was adapted for current assay.
- 24. . Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages. J. Exp. Med. 170(5), 1635–1648 (1989).
- 25. . Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182(2), 389–400 (1995).