The significance of IgM in autoimmune and infectious diseases

In this work, we first aimed to optimise the methods for recombinant antibodies production starting
from the isolation of circulating antigen-specific B cells. We aimed to produce recombinant antibodies
in their native class in order to dissect their class-specific properties. We took advantage from the
unique setting offered by the COVID-19 pandemic, which gave us the opportunity to collect blood
from donors recently infected by a novel virus and to use them to select SARS-CoV-2 spike-protein
specific B cells, applying MACACS, a technique developed in our laboratory that enables enrichment
of rare antigen-specific B cells from peripheral blood. We applied this technique to produce
recombinant antibodies against a known target and assess their functions when expressed in their native
class, including in vitro neutralisation potential, complement activation, affinity measurement and
epitope mapping. In vitro class switch of recombinant antibodies enabled the direct comparison of the
functional attributes of IgG and IgM.
In the second part of this work, we aimed to identify the target antigen of CSF IgM in MS patients. We
first screened two independent cohorts of CSF samples for CSF antibodies binding to the cell surface
of a panel of neural or glial derived cell lines. After the identification of IgM binding to a peripheral
neuroectodermal tumour (PNET) that reliably differentiates MS from controls, we applied the
optimised pipeline for cloning of SARS-CoV-2 spike-specific monoclonal antibodies to the cloning of
CSF derived PNET-binding IgM from multiple sclerosis patients, with the aim of using the
recombinantly produced IgM for antigen identification by immunoprecipitation and mass
spectrometry.
In the last part, we used the incidental detection of natalizumab in the CSF of treated patients while
looking for antibodies binding to PNET cells to develop a flow-cytometry based assay and use it to
determine natalizumab concentration in CSF, serum, and breastmilk of multiple sclerosis patients.