Rapid Identification of Diverse High-Affinity SARS-CoV-2 Antibodies

In this webinar you will learn about GenScript’s two new platforms

Beacon® single B cell screening

Precision Mutant Libraries

GenScript’s product managers (Grace Tan, PhD and Maung Win, PhD) will introduce you to the advantages of these platforms, and how they may be leveraged to expedite the discovery of antibody candidates and to optimize the properties of identified leads.

SARS-CoV-2 Antibody Discovery

Antibodies have played a critical role in the management of the COVID-19 pandemic, primarily as tools for diagnostics but also as potential therapeutics. GenScript has already developed a variety of monoclonal antibodies to the SARS-CoV-2 nucleocapsid and spike protein. This webinar will illustrate how GenScript has leveraged its Beacon® platform to expedite the discovery of neutralizing antibodies specific to the spike’s RBD.

Binding of the SARS-CoV-2 virus via its spike protein to cellular ACE2 receptors is a critical step for the fusion events leading to viral entry.

During the ongoing pandemic, tools to advance research on SARS-CoV-2 are greatly needed. However, expediting the development of reagents, such as high affinity monoclonal antibodies, is not an easy feat. Especially because most available and traditional platforms for antibody discovery rely on methods which are inherently laborious and time consuming. Such is the case of hybridoma technology, which is widely used in the development of high affinity monoclonal antibodies. Nevertheless, in the hybridoma workflow, antibody screening is a process that may not be fast tracked, taking in average 3 months for antibody selection.

Another drawback of hybridoma based methods is the inevitable loss of antibody diversity, or epitope repertoire, which occurs as the consequence of the immortalization/fusion process. Other approaches, such as display libraries, expedite the process of antibody discovery to 1-2 months at the expense of antibody quality and diversity.

Single B Cell Screening with Beacon Enabled Platform

GenScript’s newest workflow for antibody discovery takes advantage of the Beacon® B cell screening platform from Berkeley Lights. In the Beacon® Platform, optofluidic technology enables automated and high throughput screening of B cells. In Beacon®, single B cells are loaded onto a chip and sorted into individual nanopens, where they may be screened for secreted antibody specificity and function.  

The workflow starts with B cells isolated from an immunized host (e.g., spleen, lymph nodes, blood), followed by B cell enrichment and Beacon® enabled screening, which may be as short as 1 day. For antibody screening, the Beacon® platform allows for assay flexibility. Once B cells are isolated within individual nanopens, secreted antibodies may be subjected to a variety of automated assays. For example, bead based assays (e.g., using antigen or antibody coated beads) or reporter-cell based assays, where fluorescence indicates specific antibody binding. Identified candidates are sequenced, cloned, and made available for testing in small scale expression systems.

Spike-RBD Monoclonal Antibody Discovery

To ensure the highest antibody quality and diversity for screening, GenScript’s Beacon® based antibody discovery workflow relies on rabbit immunization. Additionally, to expedite B cell identification, screening relies on an antigen-specific bead assay using biotinylated RBD.

Following this workflow, GenScript’s scientists identified 142 single B cells producing antibodies specific for RBD within 24 hours. A total of 60 antibody candidates were sequenced and evaluation of top-leads showed high affinity, with KD in the range of 10-9 to 10-10 M. Additionally, compared to antibodies discovered through hybridoma technology, candidates identified through Beacon® screening showed a higher diversity in epitope recognition. Therefore, by allowing identification of high affinity RBD antibodies to a broad range of epitopes, single B cell screening is sure to facilitate downstream applications such as immunoassays where antibody pairs to a single antigen are commonly used.

SARS-CoV-2 RBD antibody pairs identified through single B cell screening. Antibody pair, BS-R2B2&BS-R2B12, was evaluated for use in Sandwich ELISA (sensitivity 20pg/mL).

Options for Antibody Optimization

Molecular engineering provides the opportunity to further improve the properties of antibody candidates identified through single B cell screening. GenScript’s Precision Mutant Libraries (PMLs) enable antibody candidate optimization by creating precise sequence changes.

What are PMLs? High quality, diverse synthetic libraries containing only desired mutants. Precision is enabled by GenScript’s next generation semiconductor-based DNA synthesis, which is coupled to an automated platform, providing control over the incorporation of individual nucleotides into the forming DNA strand on a silicon chip. PMLs provide several key advantages including:

These are advantages that set PMLs apart from traditional libraries such as degenerate mutant libraries, which are larger, and offer no control over amino acid ratio, or codon optimization.

Comparison between a degenerate peptide library and GenScript’s PML demonstrates the power of semiconductor-based technology for library creation. (Right) A PML library was designed to incorporate the following mutations: 3 amino acid in the first position (AA1), 10 amino acids in AA2, and 20 amino acids in AA3 and AA4. These precise changes are only achievable in PMLs and not in degenerate libraries, which additionally show codon over and under-representation (Left).

GenScript offers three types of PML services including site saturation, saturation scanning, and combinatorial mutant libraries. These libraries provide the user the option of mutating a single site, multiple sites, or a combination of multiple sites and regions. Libraries are built by precise synthesis of individual oligos and are offered as double stranded DNA or cloned into plasmids suitable for downstream applications. NGS is used as the main quality control analysis to evaluate the sequence profile of the resulting libraries.

Optimizing Antibody Affinity with PML

PMLs may be designed and implemented with the goal of improving the affinity of monoclonal antibodies. GenScript’s scientists leveraged the PML saturation mutagenesis platform to introduce specific mutations within various complementary determining regions (CDRs) of an antibody. Specifically, 63 sites within 6 CDRs were mutated to 19 alternate amino acids, for the generation of ~1,200 mutants. NGS based quality control assessment confirmed the even distribution of amino acid changes on all targeted sites.

Characterization of the resulting antibody candidates, through surface plasmon resonance (SPR), showed that several mutations resulted on substantially improved affinity properties.

Currently, GenScript’s scientists are leveraging the PML platform to further improve the affinities of SARS-CoV-2 RBD MonoRab antibodies as well as of single B cell derived RBD antibodies identified through the Beacon platform.

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