Development, High-Throughput Profiling, and Biopanning of a Large Phage Display Single-Domain Antibody Library

Abstract

Immunoglobulin G-based monoclonal antibodies (mAbs) have been effective in treating various diseases, but their large molecular size can limit their penetration of tissue and efficacy in multifactorial diseases, necessitating the exploration of alternative forms. In this study, we constructed a phage display library comprising single-domain antibodies (sdAbs; or "VHHs"), known for their small size and remarkable stability, using a total of 1.6 × 10⁹ lymphocytes collected from 20 different alpacas, resulting in approximately 7.16 × 10¹⁰ colonies. To assess the quality of the constructed library, next-generation sequencing-based high-throughput profiling was performed, analyzing approximately 5.65 × 10⁶ full-length VHH sequences, revealing 92% uniqueness and confirming the library's diverse composition. Systematic characterization of the library revealed multiple sdAbs with high affinity for three therapeutically relevant antigens. In conclusion, our alpaca sdAb phage display library provides a versatile resource for diagnostics and therapeutics. Furthermore, the library's vast natural VHH antibody repertoire offers insights for generating humanized synthetic sdAb libraries, further advancing sdAb-based therapeutics.

Keywords: antibody library; next-generation sequencing; phage display; single-domain antibody.

Figure 1

Schematic representation of the process of constructing an alpaca VHH library, phage display-based biopanning, and the applications of VHHs. The VHH library was constructed from 1.6 × 10⁹ alpaca lymphocytes, and VHHs were selected through phage display-based biopanning. VHHs hold promise for a wide range of applications, including display technology, diagnostics, therapeutics, and research.

Figure 2

Next-generation sequencing (NGS)-based characterization of the constructed single domain antibody library. (A) Flowchart illustrating the process of NGS analysis. (B) Redundancy analysis of the variable regions (VHHs) of heavy-chain-only antibody sequences in the constructed library. The graph displays the number of replicates (n) as a percentage of VHH sequences out of the total number of analyzed sequences.

Figure 3

Germline V and J gene pairing frequencies in the VHH repertoire of the constructed library. (A) Visualization of the germline V and J gene pairing frequencies within the VHH repertoire of the constructed library. This two-dimensional heatmap depicts the frequencies of the pairings between the three IGHV gene families and the seven IGHJ gene families. (B) Further depiction in heatmap format, showcasing the frequencies of pairings between the 63 individual IGHV gene segments and the seven IGHJ gene families within the VHH sequences.

Figure 4

The length distribution of VHHs’ complementarity-determining regions (CDRs) in the constructed library. (A–C) Frequencies of the lengths of CDR1 (A), CDR2 (B), and CDR3 (C) expressed as percentage bar graphs. The most common length of CDR1 and CDR2 was eight amino acids. The lengths of CDR3 were found to range from 5 to 31 amino acids.

Figure 5

Amino acid compositions in VHH’s CDRs and Framework 2 (FR2). (A) Comprehensive analysis of the amino acid compositions across the complete CDR1, CDR2, and CDR3 within the constructed library. (B,C) In-depth investigation into the amino acid compositions at each position within CDR1, CDR2, and CDR3. (D) Detailed analysis of the characteristic amino acid compositions at Positions 42, 49, 50, and 52 within the FR2. In all panels (A–D), the amino acid compositions within CDRs and FR2 are expressed as percentages and visualized as stacked bars in distinct colors. Each stacked bar represents the proportion of each amino acid at specific positions. The x-axis indicates the amino acids’ positions in accordance with the IMGT’s numbering.

Figure 6

The frequency of non-germline amino acids within CDR1 and CDR2 in the constructed library. The graph depicts the percentage of VHH sequences with different numbers of non-germline amino acid residues in CDR1 and CDR2. The x-axis shows the number of non-germline amino acids in CDR1 and CDR2, while the y-axis indicates the percentage of VHH sequences. The data reveals the distribution of mutations in the CDRs, with some sequences exhibiting no mutations, and others displaying varying numbers of non-germline amino acids.

Figure 7

Somatic diversification in the constructed library. (A) Variability indexes of VHHs in the constructed library and the alpaca germline database. The variability index is shown as a bar graph, with the positions exhibiting high variability being marked. (B) Mutation (substitution and insertion) hotspots in alpaca VHH genes. The frequencies of mutations at each position within the CDRs are illustrated. The x-axis indicates the amino acids’ position for each CDR, as defined by the IMGT’s numbering.

Figure 8

Position-specific amino acid frequencies of mutations at each position in the CDRs of the VHH in the constructed library. Position-specific amino acid frequencies of substitutions (A) and insertions (B) at each position in the CDRs of the variable domain in the constructed library. The x-axis indicates the amino acids’ position of each CDR, as defined by the IMGT’s numbering.