A presentation on innovative antibody discovery by Amelie Kutschera, MSc., Single B Cell Scientist, & Iwona Budnicki, Manager Cellular Immunology at Genovac
by Silvia Hnatova
At Antibody Engineering & Therapeutics Europe Virtual earlier this year, Ms. Amelie Kutschera, Single B Scientist from Genovac, presented the strategies for antibody discovery against difficult targets combining the genetic immunization and Berkely Lights Beacon®.
The traditional immunization options to discover novel antibodies include a hybridoma screening platform, through which monoclonal antibodies can be discovered in the timeframe of 2-5 weeks.
The beacon screening method developed by Genovac using genetic immunization allows the screening of antibody candidates in one day.
Ms. Kutschera explained that genetic immunization is advantageous because of the wide epitope coverage, high sensitivity and affinity and no time-consuming antigen production required.
Genovac uses a genetic immunization strategy that was previously demonstrated through the epitope coverage library to recognize specific extracellular domains of human VEGFR-2.
The technology can distinguish highly conserved molecules, for example targeting CLDN6, but not CLDN9 and vice-versa despite only 3 amino acid differences.
The strategy led to the development of therapeutics such as monoclonal antibodies for cancer therapy.
The agonistic anti-OX40 mAbs developed through genetic immunization led to tumor regression in late-stage melanoma patients and are now tested in clinical trials.
The Berkeley Lights Beacon® is now Genovac’s golden standard methodology for antibody discovery.
It allows the screening of more than 80,000 primary plasma cells per day, performing multiple assays helping to limit the number of antibodies to be screened, improving the overall screening speed.
An example of the cell-based assays that can be included downstream includes an optimized transient expression system for optimal on-chip assay performance, transiently transfected cells are sorted for high expressors, enhancing the sensitivity.
A typical Berkely Lights Beacon® run involves genetic immunization of mice followed by high-purity isolation of plasma cells. These are then imported and single-cell cloned into the 11k pens of an Optoselect Chip.
Through a beads-based assay, the IgG producing cells are identified and a cell-based assay using transfected mammalian cells as reporter systems enables the identification of target-specific plasma cells.
The variable heavy and light chain sequences are exported one by one to be PCR amplified, cloned, and expressed in mammalian cell lines for further tests.
The technology was used previously in case studies to identify antibodies against GPCR receptors, identifying an increased number of candidates and a higher specificity of antibody candidates than through a hybridoma assay.
The challenges of the platform on efficiency and speed may be complemented through the use of B cells harvested from lymph nodes, combined with single B cell platforms.
Ms. Kutschera devoted the next part of her talk to the OmniRat® derived plasma cells used for the discovery of neutralizing antibodies against SARS-Cov-2.
Using the method, it was possible to identify several potential antibodies reactive with SARS-CoV-2 spike protein and its receptor-binding domain (RBD).
Genovac is now testing fully recombinant anti-RBD antibodies. The antibodies were able to bind to ACE2 of SARS-CoV-2 when incubated with the RBD of SARS-CoV-2.
The talk was closed by presenting future directions of Genovac’s platforms, including the development of a species repertoire for the beacon strategy.
The talk was followed by an interesting Q&A session, focused on practical issues with the beacon platform.
The capacity of the beacon was discussed, with Dr. Iwona Budnicki, Manager, Cellular Immunology from Genovac specifying that it depends on the complexity of the target, which can be in the order of dozens or hundreds.
The screening advantages of the beacon were compared to the hybridoma technology, and Dr. Budnicki highlighted the direct capacity of the beacon to screen for antibody-screening cells omitting the fusion step, increasing the throughput, and the output for the antibody of the desired characteristic.
In addition, the beacon can use different types of cells, from B cells or the bone marrow, unlike the hybridoma approach.
Amelie Kutschera presents at the Antibody Engineering & Therapeutics Europe Virtual conference
The specialty of bone marrow is that these are sparse but terminally differentiated B cells with very high diversity, increasing the sequence variety of the candidate antibodies.
Dr. Budnicki further elaborated on the attractive feature of the beacon screening compared to the hybridoma approach.
One of them is the ability to carry out consecutive assays, enabling the downselection of the bead candidates to antibodies with the desired characteristics faster than the fusion approach.
Memory B cells can also be used on the beacon to screen plasmablasts (activated memory B cells). The beacon technology can be used for screening antibodies from various species, including humanized mice and rat models.
