The Antibody-Extractor software platform offers a set of algorithms exclusively dedicated to antibody sequence and repertoire analysis.

Combining phage display with NGS for the discovery of potent antibody variants

In this use case we describe a novel in-silico antibody discovery methodology called “In vitro maturation”. The methodology was successfully used by the company argenx.

Antibody discovery teams often focus on phage display-expressed antibodies only, and miss out on a significant part of the library diversity. The aim should always be to leverage the full immune library diversity potential. However, processing large numbers of clones is limited by human and technical resources.

At AbnomX we recommend a pragmatic lead generation process based on immunoinformatics that combines phage display for the selection of specific binders and NGS for the identification of binder variants.


The “in-vitro maturation process” starts with a phage display sequence analysis. The Antibody-Extractor module “Annotator” annotates the VHs CDRs and Frameworks and removes redundancy. Next, “Abaligner” performs a multiple alignment of the VHs and sorts the alignment by CDR3 length for VH family identification.

Then, one conducts a boosted immune library NGS analysis. Using “Abcluster” the VHs are clustered based on their CDR3 identity and length. This will give an overview of the highly expressed VH families, and the library diversity. Then “Abquery” screens the library with phage-display selected VHs and extracts a number of variants from the library.

Now it is time for “Hotspot” to compute the Wu-Kabat variability for all the extracted variants and highlight the potential mutation residues.

At this point the real in-vitro maturation design work can start: A random mutagenesis is performed on the identified targeted residues resulting in a library size of 2.1×109 members.


In this use case, argenx used Antibody-Extractor” to identify three variants out of 40,000 VHs. A significantly improved potency was observed for all three VH variants compared to the parental clone. Mutagenesis of the identified residues generated several VHs with up to a 6-fold improved potency compared to the phage display-selected VHs.

In conclusion, immunoinformatics can play a key role in the antibody discovery process.