Super-Resolution Imaging Approaches To Determine The Organization of B Lymphocyte Surface Receptors

by Amir Mohammad Rahimi
Cumulative thesis
Date of Examination:2025-07-02
Date of issue:2025-08-21
Advisor:Dr. Luis Felipe Opazo Dávila
Referee:Dr. Luis Felipe Opazo Dávila
Referee:Prof. Dr. Jürgen Wienands
Persistent Address: http://dx.doi.org/10.53846/goediss-11456

 

 

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Abstract

English

This thesis presents a multidisciplinary investigation into B cell membrane biology andnanobody-based molecular targeting, spanning the development of new imaging techniques,a novel nanobody discovery platform, and functional validation of therapeutic candidates.Across three chapters, I address fundamental questions in B cell receptor (BCR) signaling,present a novel nanobody discovery technique for membrane proteins, and translationallyapply these nanobodies for immunotherapy. In Chapter 1, I aim to examine a long-standingdebate regarding the nanoscale organization and activation mechanism of BCRs. To addresslimitations in imaging suspended B cells, I developed a novel “Membrane Sheet PressingMethod (MSPM)” technique to generate high-quality membrane sheets, which I combinedwith super-resolution microscopy and stoichiometric labelling using specific monovalentnanobodies. This enabled us to have an accurate estimation of BCR organization across largesurface areas. Using engineered cell lines with specific BCRs and antigens of defined valency,we demonstrated that BCR clustering size is the primary trigger for signaling, rather thanantigen binding or antigen valency alone, and that BCR-cluster size acts as a threshold foractivation. In Chapter 2, I introduce Reverse Phage Display (RPD), a method that invertstraditional nanobody selection by identifying binders to native membrane proteins withoutprior knowledge of the target. RPD overcomes challenges of high failure rate for membraneproteins, recombinant antigen production, and antigen conformation loss, and uniquelyenables the discovery of previously unknown or poorly characterized surface proteins,including those relevant to cancer, a key feature that classical display methods lack. Using thisapproach, I discovered nanobodies against CD45, surface IgM, and CD38, along with severalcandidates still under investigation. Notably, two of the selected nanobodies resulted not onlyin high specificity, but with strong affinities in the low picomolar range, highlighting theplatform’s stringency and precision. In Chapter 3, I focus on CD38 as a therapeutic target. Icharacterize NbC6, a nanobody with superior affinity and a unique epitope profile, capable ofoutperforming FDA-approved therapeutic antibodies Daratumumab and Isatuximab in keyassays. Together, in this thesis I perform nanoscale B cell membrane analysis, rapid nanobodydiscovery, and translational antibody engineering.
Keywords: Antibody; Nanobody; CD38; CD45; STED; BCR; Multiple Myeloma
Schlagwörter: CD45; CD38; BCR; Multiple Myeloma; B cell receptors