Next-generation sequencing-guided identification and reconstruction of antibody CDR combinations from phage selection outputs

Abstract

Next-generation sequencing (NGS) technologies have been employed in several phage display platforms for analyzing natural and synthetic antibody sequences and for identifying and reconstructing single-chain variable fragments (scFv) and antigen-binding fragments (Fab) not found by conventional ELISA screens. In this work, we developed an NGS-assisted antibody discovery platform by integrating phage-displayed, single-framework, synthetic Fab libraries. Due to limitations in attainable read and amplicon lengths, NGS analysis of Fab libraries and selection outputs is usually restricted to either VH or VL. Since this information alone is not sufficient for high-throughput reconstruction of Fabs, we developed a rapid and simple method for linking and sequencing all diversified CDRs in phage Fab pools. Our method resulted in a reliable and straightforward platform for converting NGS information into Fab clones. We used our NGS-assisted Fab reconstruction method to recover low-frequency rare clones from phage selection outputs. While previous studies chose rare clones for rescue based on their relative frequencies in sequencing outputs, we chose rare clones for reconstruction from less-frequent CDRH3 lengths. In some cases, reconstructed rare clones (frequency ∼0.1%) showed higher affinity and better specificity than high-frequency top clones identified by Sanger sequencing, highlighting the significance of NGS-based approaches in synthetic antibody discovery.

Figure 1.

Strategy for CDR strip generation and sequencing. ssDNA rescued from round-3 phage pools is subjected to Kunkel mutagenesis for deleting the intervening framework regions between diversified CDRs. This step links the diversified CDRs next to each other (L3-H3 from Library-S selections and L3-H1-H2-H3 from Library- F selections) without losing the CDR pairing information. The product from the mutagenesis reaction is used as a template to generate a PCR amplicon of the CDR strip. Quantified, multiplexed amplicons are subjected to emulsion PCR and Ion Torrent sequencing. L3-H3 and L3-H1-H2-H3 strips are sequenced on 200 and 400 bp chips, respectively.

Figure 2.

Strategy for reconstructing Fab clones from NGS information. CDR strips (L3-H3 or L3-H1-H2-H3) generated from round-3 phage pools are subjected to Ion Torrent Sequencing. Following NGS analysis, desired CDR combinations are reconstructed by cloning CDR-encoding oligonucleotides into the Hu4D5 Fab-encoding template phagemid by Kunkel mutagenesis.

Figure 3.

Jagged-2 Fabs from Library-S. (A) Diversified CDR sequences and phage-displayed Fab binding characteristics for seven Jagged-2 Fabs isolated by Sanger sequencing. (B) Fab yield, affinity and specificity of purified Jagged-2 Fabs. Fab yield was estimated for 1 L bacterial culture. EC₅₀ values and K_D values for Fabs binding to Jagged-2 were determined by multi-point Fab-ELISA and bio-layer interferometry, respectively. Error values represent the standard error of regression. Fab specificity was determined using single- point Fab-ELISA at 100 nM Fab concentration. Phage-ELISA and specificity-ELISA Abs₄₅₀ values are shown as heat maps. In ELISA heat maps, Notch-1/2/3 and Jagged-1/2 target proteins are indicated as N1, N2, N3, J1 and J2.

Figure 4.

NGS-assisted reconstruction of rare Fab clones from the Jagged-2 selection. (A) Enriched sequences (>1%) from the round-3 CDRL3-CDRH3 strip sequencing output. Seven Fabs isolated by Sanger sequencing are indicated with arrows. (B) CDRL3 and CDRH3 length distribution of the Jagged-2 selection output (round-3). (C) Sequence logos showing the positional amino acid composition of CDRL3 and CDRH3 sequences from the Jagged-2 selection output. The Kabat scheme was used for numbering amino acids. (D) Diversified CDR sequences and phage-displayed Fab binding characteristics for two Jagged-2 rare clones reconstructed from NGS information. (E) Fab yield, affinity and specificity of purified rare Fabs J2/S/R1 and J2/S/R2. EC₅₀ values were determined by Fab-ELISA. Error values represent the standard error of regression. Fab specificity was determined at 100 nM Fab concentration. Phage-ELISA and specificity-ELISA Abs₄₅₀ values are shown as heat maps.

Figure 5.

Notch-3 Fabs from Library-S. (A) CDRL3 and CDRH3 length distributions of the Notch-3 selection output (round-3). (B) Diversified CDR sequences and phage-displayed Fab binding characteristics for Notch-3 Fabs. (C) Fab yield, affinity and specificity of Notch-3 Fabs. EC₅₀ values were determined by Fab-ELISA. Error values represent the standard error of regression. Fab specificity was determined at 1 μM Fab concentration. Phage-ELISA and specificity-ELISA Abs₄₅₀ values are shown as heat maps. In panels (B) and (C), reconstructed rare clones are highlighted in gray.

Figure 6.

Jagged-2 Fabs from Library-F. (A) CDRL3 and CDRH3 length distribution of the Jagged-2 selection output (round-3). (B) Diversified CDR sequences and phage-displayed Fab binding characteristics of 9 top Fabs isolated by Sanger sequencing and 7 rare Fabs reconstructed from L3-H1-H2-H3 NGS information. (C) Fab yield, affinity and specificity of purified Fabs. EC₅₀ values were determined by Fab-ELISA. Error values represent the standard error of regression. Fab specificity was determined at 1 μM Fab concentration. Phage-ELISA and specificity-ELISA Abs₄₅₀ values are shown as heat maps. In panels (B) and (C), reconstructed rare clones are highlighted in gray.

Figure 7.

General strategy for strip generation with naïve libraries. Strip generation can be used to reduce the amplicon size for any NGS platform and library. Kunkel mutagenesis using a primer with regions complementary to the constant light (C_L) and upstream of the framework heavy chain 1 (FRH1) region will result in a template with reduced size for subsequent PCR amplification and NGS sequencing.