Videoconference : link.
Title : Characterization of therapeutic antibody/receptor pairs nanoscale organization & trafficking by quantitative single molecule localization microscopy combined with high content screening
SIBARITA, Jean-Baptiste, Directeur de Thèse
PERRAULT, Christelle, co-superviseure de Thèse
Defense in english
Immuno-oncology is a young and growing field at the frontier of cancer therapy. Immuno-oncology therapies aim to stimulate the body’s immune system to target and attack the tumor through therapeutic antibodies, by binding and modifying the intracellular signaling of T-cells (lymphocytes playing a central role in the immune response) surface receptors. Understanding how the spatial organization of receptors and signaling proteins is regulated and how it determines lymphocyte activation and cell fate decisions has become a ‘holy grail’ for cellular immunology. To achieve this goal, a better comprehension of antibodies functions and subcellular trafficking is requested to explain the differential efficacies of therapeutic candidates targeting receptors of interest. Quantitative super-resolution microscopy provides access to the nanoscale organization of membrane receptors playing a physiological role. It offers a new investigation tool for antibody optimization as well as maximizing their functional efficacy. In combination with high throughput screening techniques, it has the potential to play a crucial role in the early phases of projects in which it is necessary to select the best antibodies from banks that may contain several hundreds of them.
The goal of this PhD thesis was to functionally characterize receptor/antibodies pairs organization and trafficking by quantitative single-molecule localization microscopy (SMLM) combined with high content screening (HCS). In this context, we have developed and used an HCS-SMLM platform to characterize multiple antibodies targeting T-cell membrane receptors, allowing gathering unprecedented quantitative insight of potential therapeutic candidates. We also optimized the single objective light-sheet microscope (soSPIM) to permit 3D mapping of membrane receptors across an entire T-cell, with single molecule resolution. It allows 3D nanoscale imaging of T-cells in more physiological conditions, and provide complementary information compared to large scale single molecule screening experiments. Altogether, these developments improved our comprehension of antibody mode of action on receptors at the single cell level. Large-scale experiments performed during this work required the development of several software for the automation of the acquisition and the statistical analysis of the Terabytes of single molecule data generated.
This project is focused on targeting PD-1, a control point of the immune system involved in the modulation of immune cells activation. The first part of the thesis was mainly devoted to the implementation of new protocols for PD-1 receptors super-resolution imaging on activated Jurkat cells. In the second part, we further investigated the impact of known anti-PD-1 therapeutic antibodies used in clinics, on the nanoscale spatial organization and dynamics of PD-1 receptors in living cells using our HCS-SMLM platform. This work provides the proof of concept of the capacity of these cutting-edge imaging techniques to characterize quantitatively different therapeutic monoclonal antibodies targeting PD-1 on T-cell membrane.
Keywords: Immunotherapy, High Content Screening, Super-Resolution Microscopy, Single Molecule Localization Microscopy, Software Development, Datamining
NAGERL, U Valentin