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Establishing highly multiplexed microfluidic screening of protein-antibody interactions

Seah, Yu Fen Samantha

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Abstract

Antibodies play an increasingly important role in modern medicine, with monoclonal antibody sales expected to reach nearly USD 200 billion by 2024. High-throughput antibody screening is possible with phage display technologies, however, these are based on antibody binding, which does not necessarily correlate with the phenotypic effects of antibodies on target cells. Traditional functional antibody screening can also be performed with hybridoma cell technology, but as individual hybridoma cells have to be grown up into colonies and tested, this process is both time-consuming and expensive, and only a mere few thousand clones can be obtained and screened.

Droplet microfluidics has been utilised for the screening of individual antibody-secreting cells, as the small droplet volumes enable the accumulation of antibodies to a functional concentration within hours. Droplet microfluidics is also the basis of emulsion-based single-cell transcriptomic assays, where the co-encapsulation of barcoded entities with cells enables the labelling of all mRNA from an individual cell with the same barcode, permitting high-throughput single-cell transcriptomic analyses.

In this thesis, we aimed to evaluate the feasibility of an antibody screening technology that would combine these two applications of droplet microfluidics, and to set up the individual components necessary for such a technology. This technology would enable the study of the effects of single antibody-secreting cells on the transcriptomes of single target cells of interest, in order to identify antibodies of interest.

This requires the co-encapsulation of two different cell types. As this process is governed by Poisson distribution, most droplets cannot reach the desired cell occupancy. To overcome this, we have optimised a picoinjection workflow to selectively inject lysis buffer into droplets with desired cell occupancies, in order to maximise the utility of the sequencing results in our eventual screens.

In addition, we have identified appropriate model systems in which induced transcriptomic changes can be identified at a single-cell level, which can thus be used for further proof of concept and optimisation experiments. We have also evaluated the feasibility of detecting perturbations from the transcriptomic data of a small number of cells treated with drugs, as the identification of antibody hits in our future screens would require the identification of single perturbed transcriptomes in a background of untreated transcriptomes.

Furthermore, as our antibody screening technology would require a means of antibody sequencing, we have developed a Drop-seq-compatible antibody sequencing methodology that enables the identification and elucidation of heavy and light chain antibody variable region sequences. We apply this methodology to sequence mixtures of four different hybridoma cell lines mixed at different ratios and to sequence a diverse population of antibody-secreting cells, for which we have no prior knowledge of the heavy and light chain variable regions present.

These individual components pave the way for the development of a microfluidic antibody screening pipeline that employs single-cell transcriptomics. By studying gene expression changes as a proxy for the global phenotypes of target cells, the effects of different antibodies on various different targets can be simultaneously monitored, permitting highly multiplexed screening campaigns.

Item Type: Dissertation
Supervisor: Stegle, Dr. Oliver
Place of Publication: Heidelberg
Date of thesis defense: 14 January 2021
Date Deposited: 20 Jan 2021 13:30
Date: 2022
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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