Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial V_H:V_Llibraries from immunized animals

Abstract

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring

“…To further expand the utility of the Beacon platform, we explored a variety of library formats, with yeast display as a primary focus due to its wide versatility to perform protein and peptide engineering (Cherf & Cochran, ). Yeast display has been used to discover de novo binders (McMahon et al, ; Wang et al, ), affinity mature existing binders (Tiller et al, ), engineer in pH‐sensitivity (Schroter et al, ), improve protein thermal stability (Jones, Tsai, & Cochran, ), and enhance enzyme kinetics (I. Chen, Dorr, & Liu, ). Our work used yeast‐displayed peptides isolated from an affinity maturation campaign against a receptor ECD to develop a binding affinity assay on the platform.…”

Deep sequencing methods for protein engineering and design

“…Advances in pairing V H and V L sequences from individual B cells [6] allows one to identify antigen-specific antibodies directly from sequencing, including panels of antibodies targeting Ebola virus [7] and ricin [8]. Methodological details and limitations associated with identification of rare clones and evaluation of library diversity are presented in a recent review [9].…”

Integrating high‐throughput screening and sequencing for monoclonal antibody discovery and engineering

“…In addition to bulk sequencing, single‐cell sequencing that reveals native V H and V L chain pairing has also been employed for antibody discovery. For example, Wang and colleagues isolated plasmablasts from the draining lymph nodes of immunized mice and obtained V H and V L chain pairing by following the method described previously (see previous section) . After sequencing paired repertoires, it was again revealed that plasma cell diversity in lymph nodes was also polarized to a great extent.…”

“…Antibody repertoire mining by Ig‐seq and phage display were therefore found to be two complementary technologies that led to the isolation of different antigen‐specific antibodies. Conversely, following a similar study design, monoclonal antibodies were discovered by either using Ig‐seq data from lymph nodes or performing yeast display on libraries generated from bone marrow and spleen from the same immunized mouse, even resulting in clones that may have originated from the same B‐cell lineage …”