Discovering Selected Antibodies From Deep-Sequenced Phage-Display Antibody Library Using ATTILA.

Phage display is a powerful technique to select high-affinity antibodies for different purposes, including biopharmaceuticals. Next-generation sequencing (NGS) presented itself as a robust solution, making it possible to assess billions of sequences of the variable domains from selected sublibraries. Handling this process, a central difficulty is to find the selected clones. Here, we present the AutomaTed Tool For Immunoglobulin Analysis (ATTILA), a new tool to analyze and find the enriched variable domains throughout a biopanning experiment. The ATTILA is a workflow that combines publicly available tools and in-house programs and scripts to find the fold-change frequency of deeply sequenced amplicons generated from selected VH and VL domains. We analyzed the same human Fab library NGS data using ATTILA in 5 different experiments, as well as on 2 biopanning experiments regarding performance, accuracy, and output. These analyses proved to be suitable to assess library variability and to list the more enriched variable domains, as ATTILA provides a report with the amino acid sequence of each identified domain, along with its complementarity-determining regions (CDRs), germline classification, and fold change. Finally, the methods employed here demonstrated a suitable manner to combine amplicon generation and NGS data analysis to discover new monoclonal antibodies (mAbs).

Production of a recombinant Fab in Pichia pastoris from a Monocistronic expression vector.

Recombinant Fab is usually expressed using dicistronic vectors producing the heavy and light chains separately. We developed an improved vector for Fab fragment expression in Pichia pastoris, which allows a stoichiometric expression of both chains based on a monocistronic arrangement. The protein is produced as a unique polypeptide harbouring a KEX2 processing site between both chains. After KEX cleavage, a correctly folded mature Fab is formed. The produced recombinant protein is characterized as a heterodimeric functional Fab. The vector described is a new tool for the proper expression of antibody fragments or any heterodimeric polypeptides.