Investigation of Nanoscale Distance-Dependent Antibody-Antigen Binding for Mast Cell Activation

When and Where

Nov 29, 2022
9:00am - 9:15am

Hynes, Level 1, Room 104

Presenter

Leonie Schneider

Carmen Martínez-Domínguez

Kersten Rabe

Christof Niemeyer

Co-Author(s)

Leonie Schneider¹,Carmen Martínez-Domínguez¹,Kersten Rabe¹,Christof Niemeyer¹

Karlsruhe Institute of Technology¹

Abstract

Leonie Schneider¹,Carmen Martínez-Domínguez¹,Kersten Rabe¹,Christof Niemeyer¹

Karlsruhe Institute of Technology¹

Cellular immune responses in mast cells are regulated in the early stages of cell signalling and play a pivotal role in the initiation and development of type I hypersensitivity reaction. The spatial organization of IgE's high-affinity transmembrane receptor, FcεRI, into molecular clusters stimulates inflammatory mediator degranulation, cytokine production and phospholipid production.[1] Receptor-clustering is mediated by multivalent antigen binding of the IgE antigen binding fragments (Fab) of receptor-bound antibodies on the cell surface. Here, the spatial tolerance of Fabs towards repetitive nanopatterns of antigens is crucial for initiation or inhibition of an immune response.[2] To investigate structural constraints of antibodies binding to multivalent antigens to initiate effector functions and cell activation, we utilize supramolecular DNA architectures decorated with small hapten molecules and present them to the cell membrane. This Multiscale-Origami Structures as Interfaces for Cells (MOSAIC) technology allows precise control over the number, stoichiometry and arrangement of the signalling molecules on the nanometer scale, imitating the ultrastructural features of biomolecular assemblies.[3] In this contribution we report on hapten-decorated DNA origami nanostructures (DON) to investigate the spatial tolerance of free and membrane-associated antibodies on the nanoscale regarding antigen-mediated effects on mast cell activation. We designed distinctive nanoscale arrangements of haptens to induce receptor clustering and for the first time investigated the relation between antigen-binding affinity and mast cell signalling, an important feature for tailoring next generation monoclonal antibodies (mAb) for immunotherapy. We synthesized rectangular DON with a size of 70 x 100 nm2 as molecular pegboards and incorporated protruding oligonucleotides for DNA-directed immobilization on solid surfaces and dinitrophenol (DNP)-ligands with precisely defined distances. We investigated the interaction kinetics of anti-DNP IgE and anti-DNP IgG to these DNP-decorated DON with distinct DNP-distances of 6 – 30 nm aiming for a better understanding of the native structure and Fab flexibility of antibodies. We also investigated the binding kinetics of DNP-decorated DON and FcεRI-receptor-bound anti-DNP IgE antibodies on the surface of rat basophilic leukemia (RBL)-2H3 cells, a model for mast cells, using LigandTracer technology.[4] To put our results in a biological context, we performed cell signalling assays to study the effect of antigen-antibody binding affinity and receptor-clustering on cellular activation.[5] To the best of our knowledge, these studies are the first to investigate the distance dependence of free and receptor-bound IgE antibodies using surface-immobilized hapten-decorated DNA origami to elucidate fundamental biological principles of mast cell activation. [1] D. Sil, J. B. Lee, D. Luo, D. Holowka, B. Baird, ACS Chemical Biology 2007, 2, 674-684. [2] A. Shaw, I. T. Hoffecker, I. Smyrlaki, J. Rosa, A. Grevys, D. Bratlie, I. Sandlie, T. E. Michaelsen, J. T. Andersen, B. Hogberg, Nat Nanotechnol 2019, 14, 184-190. [3] A. Angelin, S. Weigel, R. Garrecht, R. Meyer, J. Bauer, R. K. Kumar, M. Hirtz, C. M. Niemeyer, Angew Chem Int Ed Engl 2015, 54, 15813-15817. [4] S. Bondza, A. Marosan, S. Kara, J. Lösing, M. Peipp, F. Nimmerjahn, J. Buijs, A. Lux, Front Immunol 2021, 11, 609941-609941. [5] L. Schneider, C. Martínez-Domínguez, K. S. Rabe, C. M. Niemeyer, 2022, in preparation.