Facile isolation of high-affinity nanobodies from synthetic libraries using CDR-swapping mutagenesis

Abstract

The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of low-affinity variants during the sorting of yeast-displayed libraries to directly isolate high-affinity nanobodies without the need for lead isolation and optimization. We expect this approach, which we demonstrate for SARS-CoV-2 neutralizing nanobodies, will simplify the generation of high-affinity nanobodies. For complete details on the use and execution of this profile, please refer to Zupancic et al. (2021).

Keywords: Antibody; Biotechnology and bioengineering; Cell Biology; Flow Cytometry/Mass Cytometry; Molecular Biology; Protein Biochemistry; Protein expression and purification.

Graphical abstract

Figure 1

Schematic illustration of the nanobody selection process using yeast surface display and CDR-swapping mutagenesis Step 1: A common framework nanobody library is prepared in a plasmid which enables nanobody display on yeast through a linker to the Aga2 protein. Magnetic-activated cell sorting (MACS) is first performed to enrich the naïve library for variants that bind the antigen. The enriched library is then sorted using fluorescence-activated cell sorting (FACS) to obtain a diverse population of cells that demonstrate antigen binding (see Figure 3 for details). Step 2: Nanobody plasmid DNA is isolated from yeast cells collected in the final sort performed in Step 1. PCRs are performed to amplify individual CDRs and overlapping DNA sequences from the nanobody framework and surrounding plasmid (see Figure 2 for details). DNA is then reassembled using overlap PCR to produce DNA sequences encoding entire nanobody genes composed of CDR sequences from one or more parental nanobodies. CDR-swapped nanobody library DNA is transformed into yeast cells to produce the CDR-swapped sub-library via homologous recombination in yeast. Step 3: The CDR-swapped library is displayed on the yeast surface and further sorted by FACS to select high-affinity variants (see Figure 3 for details). Plasmid DNA from yeast cells collected in the terminal sort is isolated, and the sequences of individual clones are determined. Individual nanobodies are cloned as Fc-fusion proteins and expressed via transient transfection in mammalian cells. The affinity and activity of nanobody-Fc fusion proteins is then analyzed.

Figure 2

CDR-swapping mutagenesis randomly combines CDRs from different nanobodies by individually amplifying each CDR using common framework sites and then recombining the DNA segments using overlap extension PCR (A) Protein sequence of a representative nanobody from a common framework library (McMahon et al., 2018). Framework regions are shown in black. Variable sequences of the complementarity-determining regions (CDRs), namely CDR1 (red), CDR2 (blue), and CDR3 (green), are shown as Xs. The nanobody library used in this work incorporates variation in length of CDR3. A representative length of CDR3 is shown. CDR swapping can be used to shuffle CDR3 segments of different lengths between nanobodies. (B) The DNA sequence of the nanobody and surrounding plasmid that are used for CDR-swapping mutagenesis. Gray sequences encode homologous sequences to the plasmid at the 5′ and 3‘ ends. Nanobody framework regions are shown in black. Variable region DNA sequence corresponding to CDR1 (red), CDR2 (blue), and CDR3 (green) are shown as Xs. Individual PCRs amplify CDR1 (Forward and Reverse primer #1), CDR2 (Forward and Reverse primer #2), and CDR3 (Forward and Reverse primer #3). Regions recognized by primers that amplify individual CDRs are highlighted in yellow. Finally, the DNA segments are recombined using overlap extension PCR. Plasmid DNA at the 5’ and 3′ ends is amplified, including ∼50–60 base pairs that flank the restriction sites used for vector digest, in order to prepare plasmid DNA for homologous recombination and transformation into yeast. Restriction sites (NheI and XhoI) are highlighted in purple.

Figure 3

FACS selections incorporating CDR-swapping mutagenesis enable rapid selection of high-affinity nanobodies The nanobody library is sorted first using MACS (e.g., one MACS sort against monovalent SARS-CoV-2 receptor binding domain, RBD). Next, the enriched library is sorted by FACS against bivalent antigen (e.g., RBD-Fc) at relatively high antigen concentrations, and then it is further sorted by FACS against monovalent antigens (e.g., monovalent RBD) and/or at reduced antigen concentrations. A diagonal gate is drawn during FACS selections to collect yeast cells that bind antigen in a manner that is proportional to nanobody expression to enrich for high-affinity clones. The gates are drawn to minimize the percentage of cells appearing within the gates in the control sample to avoid enrichment of nanobodies that bind the secondary reagents. After several rounds of enriching the library via FACS, yeast plasmid DNA is isolated and CDR-swapping mutagenesis is performed. Finally, additional FACS sorts are performed following CDR-swapping mutagenesis to isolate high-affinity nanobodies.