by David Orchard-Webb
By David Orchard-Webb
At the virtual Antibody Engineering & Therapeutics conference in December 2020, James Crowe Jr., Director at Vanderbilt Vaccine Center, discussed single cell technologies for the isolation of human monoclonals, the molecular basis for SARS-CoV-2 neutralization, and antibody synergistic exploration. Peripheral blood mononuclear cells (PBMCs) from survivors of COVID-19 disease were the starting material for this work.
The project started from a DARPA pandemic prevention platform. The original aspirational goal was to go from an outbreak to a cure in about 60 days. The New England Journal of Medicine released a case report on January 19, 2020 of the first contemporary COVID-19 patient in the US. Seven days later in Nashville, Dr. Crowe Jr. had received the first sample from that individual.
Chinese scientists released sequences very early, allowing Jason McLellan of the University of Texas at Austin and others to determine the structure of the Spike protein. Building on that structural data, Dr. Crowe’s team made antigens expressed in mammalian cell lines. They typically used antigen-coated beads or sometimes only the soluble antigens for antibody binding screens. Recombinant humanized FLAG-ACE2 was used for receptor blocking assays. By iterations of this, they were able to find antigen-specific B cells.
They used two different technologies in parallel to maximize chances of success; one being the sequence based 10x Genomics single cell platform, the other based on cell sorting by microfluidics and labelling with antibodies, a kind of specialized FACS, called Beacon, made by Berkeley Lights, Inc. The resulting B cells were sequenced. Dr. Crowe detailed the Berkeley Lights Beacon approach in the talk. The antibody sequences were cloned and synthesized by Twist Biosciences, ready for transfection by the Crowe lab.
The Crowe lab developed a novel single cell surrogate virus neutralization assay using the Beacon device, memory B cells, and combinations of the RBD virus antigen and ACE2 for blocking. The lab also developed a rapid real-time virus neutralization assay using an xCELLigence impedance device from ACEA Biosciences (now part of Agilent). Cytopathic effect (CPE) in a cell monolayer is detected as a loss of impedance. A chimaeric vesicular stomatitis virus/ SARS-CoV-2 hybrid was used for this purpose. Neutralizing antibodies prevent this loss of monolayer integrity and maintain impedance at control levels.
Therefore, by these two different methods they could determine neutralizing antibodies. Working with Mike Diamond lab at WUSTL, they tested neutralization against authentic SARS-CoV-2 virus and some of those antibodies were neutralizing with IC50 values close to 10-15 ng/ml.
They worked with the Baric and Diamond labs to make several different mouse models. For example, an adenovirus transduced model where humanized ACE2 was introduced into a mouse. Using anti-ifnar antibodies, virus, and treatment antibodies, they could protect against weight loss, and histopathology with a monotherapy of some of their antibodies. Other models were explored that also gave positive ‘go’ signals.
The selected antibody clones were passed on to five different companies. At the time of the presentation, the furthest along was their partner, AstraZeneca. A phase I started in August with the AZD7442 product and then it was announced in October that they were rolling out a phase III. Go to clinicaltrials.gov to see the various formats of these trials. The timeline from discovery to production at scale is remarkably rapid, even though it did not quite hit the aspiration of 60 days.
Moving forward...
Moving forward, the Crowe lab are participating in a program called AHEAD100, which aims to make antibodies for the 100 most likely causes of the future pandemics. Dr. Crowe believes that antibodies are a big part of the future of epidemic control and COVID-19 is the first real pandemic where antibodies were brought to bear. He concluded that the faster they get at making antibodies and the farther ahead they plan, the more likely antibodies will be useful very early in epidemics.
Find out more about the 2020 Antibody Engineering & Therapeutics virtual event in our full overview here.
