Quality-controlled Characterization of a Monoclonal Antibody Specific to an EC5-domain of Human Desmoglein 3 for Pemphigus Research

Overview

Journal Front Immunol

Specialty Allergy & Immunology

Date 2024 Oct 25

PMID 39450179

Authors

Rudiger Eming

Shafaq Riaz

Eliane J Muller

Anna Zakrzewicz

Uwe Linne

Ritva Tikkanen

Christine Lea Zimmer

Christoph Hudemann

Affiliations

Soon will be listed here.

Abstract

Background: Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease caused mainly by IgG autoantibodies (auto-abs) against the cadherin-type adhesion molecules desmoglein (Dsg) 1 and 3. Pathogenic anti-Dsg3 auto-abs bind to different Dsg3 epitopes, leading, among others, to signalling that is involved in pathogenic events, such as Dsg3 depletion. As central tools in research on PV, a limited number of antibodies such as AK23 are frequently used by the autoimmune bullous disease community.

Methods: Previously, we have introduced a novel Dsg3 EC5-binding antibody termed 2G4 that may potentially serve as a superior tool for numerous PV related analysis. The purpose of this study was to develop a quality-controlled production and verification process that allows I) a continuous quality improvement, and II) a verified and comprehensible overall quality with regard to pathogenic antigen-specific binding in a variety of pemphigus assays for each batch production.

Results: Thus, a workflow based on a standardized operating procedure was established. This includes the verification of purity and binding capacity (SDS-page, direct and indirect immunofluorescence) as primary parameters, and size analysis by mass-spectrometry and pathogenicity by monolayer dissociation assay.

Conclusion: We here present an extensive point-by-point quality controlled IgG production protocol, which will serve as a basis for a standardized antibody assessment in PV research.

References

Amagai M, Stanley J . Desmoglein as a target in skin disease and beyond. J Invest Dermatol. 2011; 132(3 Pt 2):776-84. PMC: 3279627. DOI: 10.1038/jid.2011.390. View

Uhlen M, Bandrowski A, Carr S, Edwards A, Ellenberg J, Lundberg E . A proposal for validation of antibodies. Nat Methods. 2016; 13(10):823-7. PMC: 10335836. DOI: 10.1038/nmeth.3995. View

Tsunoda K, Ota T, Aoki M, Yamada T, Nagai T, Nakagawa T . Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3. J Immunol. 2003; 170(4):2170-8. DOI: 10.4049/jimmunol.170.4.2170. View

Pollmann R, Schmidt T, Eming R, Hertl M . Pemphigus: a Comprehensive Review on Pathogenesis, Clinical Presentation and Novel Therapeutic Approaches. Clin Rev Allergy Immunol. 2018; 54(1):1-25. DOI: 10.1007/s12016-017-8662-z. View

Hudemann C, Exner Y, Pollmann R, Schneider K, Zakrzewicz A, Feldhoff S . IgG against the Membrane-Proximal Portion of the Desmoglein 3 Ectodomain Induces Loss of Keratinocyte Adhesion, a Hallmark in Pemphigus Vulgaris. J Invest Dermatol. 2022; 143(2):254-263.e3. DOI: 10.1016/j.jid.2022.07.030. View

Ohyama B, Nishifuji K, Chan P, Kawaguchi A, Yamashita T, Ishii N . Epitope spreading is rarely found in pemphigus vulgaris by large-scale longitudinal study using desmoglein 2-based swapped molecules. J Invest Dermatol. 2012; 132(4):1158-68. DOI: 10.1038/jid.2011.448. View

Ganeshan R, Chen J, Koch P . Mouse models for blistering skin disorders. Dermatol Res Pract. 2010; 2010:584353. PMC: 2879910. DOI: 10.1155/2010/584353. View

Hudemann C, Hoffmann J, Schmidt E, Hertl M, Eming R . T Regulatory Cell-Associated Tolerance Induction by High-Dose Immunoglobulins in an HLA-Transgenic Mouse Model of Pemphigus. Cells. 2023; 12(9). PMC: 10177252. DOI: 10.3390/cells12091340. View

van Beek N, Zillikens D, Schmidt E . Diagnosis of autoimmune bullous diseases. J Dtsch Dermatol Ges. 2018; 16(9):1077-1091. DOI: 10.1111/ddg.13637. View

10.

Zakrzewicz A, Wurth C, Beckert B, Feldhoff S, Vanderheyden K, Foss S . Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?. Cells. 2022; 11(6). PMC: 8946243. DOI: 10.3390/cells11060942. View

11.

Di Zenzo G, Amber K, Sayar B, Muller E, Borradori L . Immune response in pemphigus and beyond: progresses and emerging concepts. Semin Immunopathol. 2015; 38(1):57-74. DOI: 10.1007/s00281-015-0541-1. View

12.

Egami S, Watanabe T, Fukushima-Nomura A, Nomura H, Takahashi H, Yamagami J . Desmoglein-Specific B-Cell-Targeted Single-Cell Analysis Revealing Unique Gene Regulation in Patients with Pemphigus. J Invest Dermatol. 2023; 143(10):1919-1928.e16. DOI: 10.1016/j.jid.2023.03.1661. View

13.

Moody M, Haynes B . Antigen-specific B cell detection reagents: use and quality control. Cytometry A. 2008; 73(11):1086-92. PMC: 3105373. DOI: 10.1002/cyto.a.20599. View

14.

Kalantari-Dehaghi M, Anhalt G, Camilleri M, Chernyavsky A, Chun S, Felgner P . Pemphigus vulgaris autoantibody profiling by proteomic technique. PLoS One. 2013; 8(3):e57587. PMC: 3591405. DOI: 10.1371/journal.pone.0057587. View

15.

Kowalczyk A, Green K . Structure, function, and regulation of desmosomes. Prog Mol Biol Transl Sci. 2013; 116:95-118. PMC: 4336551. DOI: 10.1016/B978-0-12-394311-8.00005-4. View

16.

Sardana K, Garg V, Agarwal P . Is there an emergent need to modify the desmoglein compensation theory in pemphigus on the basis of Dsg ELISA data and alternative pathogenic mechanisms?. Br J Dermatol. 2012; 168(3):669-74. DOI: 10.1111/bjd.12012. View

17.

Rafei D, Muller R, Ishii N, Llamazares M, Hashimoto T, Hertl M . IgG autoantibodies against desmocollin 3 in pemphigus sera induce loss of keratinocyte adhesion. Am J Pathol. 2011; 178(2):718-23. PMC: 3069870. DOI: 10.1016/j.ajpath.2010.10.016. View

18.

Mueller E, Rahimi S, Sauta P, Shojaeian T, Durrer L, Quinche S . Standardized Production of Anti-Desmoglein 3 Antibody AK23 for Translational Pemphigus Vulgaris Research. Curr Protoc. 2024; 4(8):e1118. DOI: 10.1002/cpz1.1118. View

19.

Ma H, OFagain C, OKennedy R . Antibody stability: A key to performance - Analysis, influences and improvement. Biochimie. 2020; 177:213-225. DOI: 10.1016/j.biochi.2020.08.019. View

20.

Ducancel F, Muller B . Molecular engineering of antibodies for therapeutic and diagnostic purposes. MAbs. 2012; 4(4):445-57. PMC: 3499339. DOI: 10.4161/mabs.20776. View