Abstract: PO1471
Generation of Anti-THSD7A Antibodies Using a Human Antibody Phage Display Library
Session Information
- Glomerular Diseases: Immunology and Inflammation in IgANP, C3GP, TMA, and Nephrotic Diseases
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Köllner, Sarah, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Seifert, Larissa, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Heine, Philip Alexander / A, Technische Universitat Braunschweig, Braunschweig, Niedersachsen, Germany
- Hust, Michael, Technische Universitat Braunschweig, Braunschweig, Niedersachsen, Germany
- Tomas, Nicola M., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
Background
Membranous nephropathy (MN) is an antibody-mediated autoimmune renal disease. In the majority of cases, autoantibodies target podocyte membrane proteins such as PLA2R1 and THSD7A. We could previously demonstrate that autoantibodies in THSD7A-associated MN cause disease and recognize several extracellular domains of the target antigen. However, it remains unclear how this antibody polyreactivity relates to disease pathogenicity. The aim of this study was the generation of anti-THSD7A antibodies using a human antibody phage display library for further diagnostic and pathomechanistic studies.
Methods
In the antibody phage display technology, antibody fragments are displayed on the phage particle and the corresponding gene fragment encoding the antibody fragment is packaged in the phage particle. A selection process called panning enables the selection of antibody fragments against virtually any target structure in vitro. In this project, a naïve antibody gene library was used to select binders to the extracellular part of human and mouse THSD7A. Binders were sequenced, cloned into scFv-Fc format - an IgG like format - and produced in HEK293 cells. Binding of scFv-Fc to human and mouse THSD7A was investigated using Western blot, ELISA and indirect immunofluorescence testing (IFT). Epitope regions were mapped using an ELISA.
Results
Four binders (SAK79-B1, SAK78-C12, SAK78-E6, SAK78-F8) could be obtained. While SAK78-F8 exclusively bound human THSD7A, the other binders reacted with both human and murine THSD7A in Western blot, ELISA and IFT. A domain-specific ELISA revealed binding of SAK78-C12, SAK78-E6 and SAK78-F8 to the regions d8_d9, d1_d2 and d6_d7, respectively. SAK79-B1 did not react with any domain combination, but showed strong binding to murine and human d1_d21. We suspect a conformational epitope that is not conserved in the coated domain fragments. SAK78-C12 and SAK78-E6 were succesfully cloned into human IgG subclass backbones IgG 1-4.
Conclusion
Antibody phage display represents a powerful method to generate recombinant antibodies. The antibodies selected in our study show different binding characteristics in vitro. In vivo binding characteristics, pathogenicity and potential as a diagnostic tool, e.g. to stain THSD7A in patient biopsies, need to be determined.
Funding
- Government Support – Non-U.S.