Abstract
Purpose
Protein aggregates have been discussed as a potential risk factor related to immunogenicity. Here we developed a novel human IgG transgenic (tg) mouse system expressing a mini-repertoire of human IgG1 antibodies (Abs) for the assessment of immunogenic properties of human mAb preparations.
Methods
Transgenic mice were generated using germline versions of the human Ig heavy chain γ1 (IgH-γ1), and the human Ig light chain (IgL) κ and λ genes. Only the soluble form of human IgH-γ1 was used to avoid expression of the membrane Ig-H chain and concomitant allelic exclusion of endogenous murine Ig genes. IgG1 aggregates were generated by different stress conditions such as process-related, low pH and exposure to artificial light.
Results
The expression of human Ig proteins induced immunological tolerance to a broad range of human IgG1 molecules in the tg mice. Immunization with IgG1 aggregates demonstrated that soluble oligomers induced by significant light-exposure and carrying neo-epitopes induced a strong immune response in tg mice. In contrast, Ab aggregates alone and monomers with neo-epitopes were not immunogenic.
Conclusion
This mouse model is able to recognize immunogenic modifications of human IgG1. While the degree of stress-induced aggregation varies for different mAbs, our findings using a particular mAb (mAb1) demonstrate that non-covalently modified aggregates do not break tolerance, contrary to widely held opinion. The immunogenic potential of soluble aggregates of human IgG strongly depends on the presence of neo-epitopes resulting from harsh stress conditions, i.e. extensive exposure to artificial light.
Abbreviations
- Ab:
-
Antibody
- ADA:
-
Anti-drug-antibodies
- AFCs:
-
Antibody forming cells
- APC:
-
Antigen-presenting cells
- BM:
-
Bone-marrow
- DCs:
-
Dendritic cells
- FCM:
-
Flow cytometry
- h:
-
Human
- H:
-
Ig heavy chain
- HMW:
-
Higher molecular weight
- IC:
-
Immune complex
- Ig:
-
Immunoglobulin
- KLH:
-
Keyhole limpet hemocyanin
- L:
-
Ig light chain
- LC/MS:
-
Liquid-chromatography/mass spectrometry
- mAb(s):
-
Monoclonal antibody(ies)
- MAPPs:
-
MHC-associated peptide proteomics
- met:
-
Methionine
- MZ:
-
Marginal zone
- s.c.:
-
Subcutaneous(ly)
- SEC:
-
Size exclusion chromatography
- tg:
-
Transgen(ic)
- V:
-
Variable region
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Acknowledgments and Disclosures
We thank Y. Lang for microinjecting the transgenic constructs, R. Paul for the generation and isolation of stressed antibody fractions, G. Steiner and V.A. Iglesias for statistical analysis, J. Marty, L. Petersen, U. Nelböck, D. Schlatter, A. Bathke, J. Wendler and R. Gruebel for their experimental support and H. Hinton and P. Barrow for carefully reading the manuscript. We also thank M. Hennig and T. Schreitmueller for their generous and continued support. This work was funded by F. Hoffmann-La Roche Ltd. All authors, except A. Rolink, are employees of F. Hoffmann-La Roche Ltd.
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Bessa, J., Boeckle, S., Beck, H. et al. The Immunogenicity of Antibody Aggregates in a Novel Transgenic Mouse Model. Pharm Res 32, 2344–2359 (2015). https://doi.org/10.1007/s11095-015-1627-0
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DOI: https://doi.org/10.1007/s11095-015-1627-0