Using Reptor for NGS-enhanced antibody discovery
Bulk sequencing of the immune repertoire with ReptorTM provides a method for identifying relatives of hits with varying affinity and developability attributes. In this blog post we look at the opportunities for NGS-enhanced antibody discovery by analyzing antibody hits discovered by Alicanto® in the context of their CDR3 families.
Clonal lineages guide antibody engineering and design
We identified 70 heavy chain sequences in the repertoire with the identical CDR3. While the validated candidate was only observed in bone marrow, other members of the lineage were identified in spleen or peripheral blood mononuclear cells (PBMCs). Members of the lineage ranged in abundance from 2 reads to 148 reads (our validated candidate had abundance 7).
An anchored lineage constructed from of a subset of the 70 sequences is shown on the right, with the location of the validated heavy chain sequence starred (at the bottom of the lineage).
We selected the nearest clade to our hit in order to determine sequence logos for the CDRs and flanking sequences (shown at bottom of figure). Interestingly, within the clade we see greater variability in the CDR1 (three positions) versus only two positions in the CDR2. By analyzing the conserved and variable positions, it may be possible to identify target-binding residues  or design a rational variant library for screening .
Avoiding developability flags in candidate selection
- Red squares indicate an un-paired cysteine in the variable region.
- Red circles indicate a variable region charge greater than our hit, an attribute associated with faster non-specific clearance .
The lineage on the left is constructed on HCAbs with identical CDR3s. However, the CDR3 is a member of a collection of 143 highly similar CDR3s (CDR3 cluster). The sequence motif shows the positions that vary across the CDR3 cluster.
Our NGS-enhanced antibody discovery workflow can add value to your antibody discovery campaign. Read more about our unique Alicanto® platform for antibody discovery from serum, or our high-throughput hybridoma sequencing service with ReptorTM.
Sharma, Vikas K., et al. “In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability.” Proceedings of the National Academy of Sciences 111.52 (2014): 18601-18606. (PubMed)
Ramirez‐Benitez, Maria del Carmen, and Juan Carlos Almagro. “Analysis of antibodies of known structure suggests a lack of correspondence between the residues in contact with the antigen and those modified by somatic hypermutation.” Proteins: Structure, Function, and Bioinformatics 45.3 (2001): 199-206. (PubMed)
Nimrod, Guy, et al. “Computational design of epitope-specific functional antibodies.” Cell reports 25.8 (2018): 2121-2131. (PubMed)