"Identification of human monoclonal antibodies from single cells: two different approaches in the context of infectious diseases"

In the age of personalised medicine, creating and producing molecules with strong affinity and specificity, like monoclonal Antibodies (mAbs), presents a great research opportunity. Various techniques exist for large-scale mAb production, making them one of the most powerful tools both for research and therapy. However, the identification of monoclonal antibodies with the desired characteristics of affinity, functional activity and developability still remains a challenging task for scientists. In this context, single B cell isolation methods have become a crucial research technique to interrogate the immune repertoire in humans. This thesis aimed to investigate the feasibility, efficiency and efficacy of two distinct methods to generate monoclonal antibodies from single B cells in the context of infectious diseases. Study A was carried out at Toscana Life Sciences (TLS) in the HARD Lab group, with the support of the University of Siena while, Study B was carried out at the Department of Biochemistry of Oxford University, in the Draper Lab group. The objective of study A was to establish a rapid and efficient workflow for the generation of human recombinant monoclonal antibodies using an innovative Fluorescence Activated Cell Sorting (FACS)-free technology based on Ferrofluid particles. Starting from single Antibody Secreting Cells (ASCs), enriched for the marker CD138 by magnetic cell sorting, cells were selected for their antigen-specificity by Enzyme-Linked ImmunoSorbent Assay (ELISA) of their cell culture supernatants. These cells were then used to generate recombinant mAbs by direct transfection of PCR fragments called “minigenes”, containing variable regions of the mAbs, which were validated by ELISA for specificity to the antigen of interest. In the context of the pandemic situation, the SARS-CoV-2 infection was considered a good opportunity to test the feasibility of generating mAbs using this platform, starting from the peripheral blood of convalescent COVID-19 donors who had recovered from the disease. In this proof-of-concept study, we aimed to develop a method and demonstrate the feasibility of this in less than 10 days, while pooling a panel of SARS-CoV-2 Spike-specific mAbs for further characterisation of functional activity by neutralisation assays. The aim of study B was to isolate antimalarial human mAbs against the Plasmodium falciparum Reticulocyte-binding protein Homolog 5 (RH5) antigen. RH5 is currently a leading blood-stage malaria vaccine target and clinical trials of vaccines containing this antigen are ongoing. It plays an essential role in parasite invasion of erythrocytes as part of the PCRCR invasion complex and, through its interaction with basigin, it is a major target of growth inhibitory antibodies. Immunity directed against the blood-stage is unique because it would allow for the development of naturally-acquired immunity and reduce morbidity and mortality. However, antibody-mediated immunity against the blood stage requires high levels of antibodies, which has been difficult to achieve thus far. Starting with PBMCs samples from RH5-vaccinated volunteers, antigen-specific memory B cells were single-cell sorted by FACS using fluorescently-labelled RH5 antigen probes. While a large number of RH5-specific antibodies have been generated previously by the Draper group, the focus of this part of the thesis was to isolate human mAbs that bind to uncharacterised regions on RH5, potentially identifying new sites of vulnerability on RH5 or “inert” sites that could be excluded from RH5 in next-generation vaccine designs to improve the response. Here we have developed a repertoire of mAbs from the peripheral blood of UK adults vaccinated with PfRH5 (VAC063 trial), a vaccine reported to be safe and immunogenic in phase I/IIa (PfRH5 in adjuvant VAC063 clinical trial, NCT02927145). We generated two new panels of mAbs: one against the full-length RH5 protein (RH5.1) and one against the N-terminus portion of the protein (RH5-NT). The mAbs in the second panel will also be used to investigate the biological role of the N-terminus, which is still unclear. Both panels of mAbs were characterised by ELISA and an in vitro parasite Growth Inhibition Assay (GIA). Although both projects have common aspects, the first study is more focused on establishing a methodology to develop mAbs in a rapid and reliable way, while the second study uses an established antibody discovery pipeline and aims to develop and characterise two panels of mAbs and provide more information in the context of vaccine design.