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

Abstract

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).

Keywords: Phage display; antibody variable domains; biopanning; combinatorial library; next-generation sequencing.

Figure 1.

The ATTILA workflow. It retrieves selected VH and VL domain sequences from phage-display experiments. The ATTILA workflow reads antibody phage-display NGS sequencing, either single-end or paired-end sequence data in FASTQ format, and delivers a report of the most enriched VH and VL sequences after panning. Those marked A and B represent workflow steps focused on the following figures. ATTILA indicates AutomaTed Tool For Immunoglobulin Analysis; NGS, next-generation sequencing.

Figure 2.

The VH gene usage of raw input sequences and rearranged VDJ after translation and pattern detection. Raw VH domain sequences were analyzed after the sequence filtering step (marked as A in Figure 1) and labeled as Input, and after rearranged VDJ pattern detection (marked as B in Figure 1). The VH gene usage was assigned by Blast against a Kabat VH germline database and is shown in proportional stacked bars for 5 independent libraries’ $(R_0)$ sequencing events. Number on the right represents the total number of sequences from A and B workflow steps in Figure 1.

Figure 3.

Accessing the diversity of the antibody phage library. The antibody phage-display library was independently sampled 5 times. (A) The UpSet plot of intersection between $R_0$ sequence sets. The horizontal bar chart indicates the total number of sequences in each $R_0$. The upper bar chart indicates the intersection size between sets of sequences in one or more $R_0$. Dots represent which $R_0$ contribute to each intersection. (B) Bar chart resuming groups of intersections. Each bar indicates the sum of similar intersections’ size, as captioned in the UpSet plot.

Figure 4.

The VH sequences are selected during panning experiments. The evolution of representative sequence contents is shown during selection steps ($R_0$, $R_2$, $R_3$, and $R_4$) for panning experiment number 1 (A, C), and along $R_0$ and $R_4$ steps for 2 independent elution protocols for panning experiment 2 (B, D). In (A), the fractions of the most frequent sequences found in $R_0$ (red) and R₄ (black) are tracked along the selection process. The quantity of each sequence is plotted as the percentage of total sequences in the VDJ subset. In (B), the quantity of the most representative sequences in experiment 2 is shown for $R_0$ (central slot), $R_4$ of acid-eluted phages (left slot), and $R_4$ for peptide-eluted phages (right slot). $R_0$ overrepresented sequences are shown in red, and enriched sequences found in both elution protocols are labeled in colors. (C, D) The VH gene usage of the corresponding VDJ subsets, suggesting that the selection leads to VH gene usage bias.

Figure 5.

Enrichment of VH domain sequences observed in the panning experiments. The fold change of the 50 most enriched clones as predicted with ATTILA are plotted in descending order: (A) panning experiment 1; (B) panning experiment 2 with peptide elution; and (C) panning experiment number 2 with acid elution.