Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing

Abstract

Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day.

Keywords: Bordetella pertussis; TROP2; antibody; antibody engineering; conformational epitope mapping; epitope mapping; protein-protein interaction; tumor necrosis factor (TNF).

FIGURE 1.

Schematic of streamlined conformational epitope mapping process. 1, SSM libraries were made for 250–300-nucleotide contiguous sections along the gene of interest. Each library contains mutations in a different section of the gene. 2, sorting conditions were determined such that there was a higher probability of capturing stronger binding cells. 3, yeast libraries were labeled with biotinylated Fab and sorted by FACS. Three different gates are drawn: a gate on light scattering parameters SSC/FSC (top; unselected population), a gate set on FSC and fluorescence channel corresponding to display of antigen (middle; displayed population), and a binding gate that collects the top 5–10% of cells by fluorescence corresponding to channel for bound antibody (bottom; bound population). 4, DNA from each population was extracted and prepared for deep sequencing on an Illumina platform. The frequency of each variant in the bound and displayed populations is compared against the unselected population and used to calculate a fitness metric. For each residue, sequence entropy (bottom) for bound (black) and displayed (green) sorts is used to determine the degree of conservation. 5, sequence entropy is used to identify conserved and non-conserved residues that are used to determine the conformational epitope (orange).

FIGURE 2.

TNF-infliximab conformational epitope determination. a, a subset (41/177 residues) of the fitness-metric heat map for bound population of the TNF-inflix_scFv interaction. Sequence entropy for the display (green) and bound population (black) is plotted below with their respective cut-offs (dashed lines). b, subtractive sequence entropy analysis for TNF-infliximab interaction. Conserved residues (orange) are found mainly within the binding footprint of the TNF-infliximab interaction (cyan). Non-conserved residues (purple) can also be mapped onto structure and fall outside of the footprint (middle). These non-conserved residues can be used to find regions where false positive conserved residues appear. For clarity, only one TNF monomer is shown. c, close-up view of the structural interface between TNF (ribbon) and infliximab (cyan surface). TNF residues are colored according to sequence conservation as in panel b.

FIGURE 3.

A soluble version of the pertussis toxin S1 subunit can be expressed in E. coli and retains affinity for hu1B7. Truncated S1 in a pAK400 expression vector was produced in BL21(DE3), harvested by osmotic shock, and purified by immobilized metal affinity chromatography and size exclusion. a, SDS-PAGE of truncated S1 (S1-220K, 25.9 kDa) and full-length PTx (26.1 kDa). b, Western blot of S1-220K and PTx, probed by hu1B7 and GαhFc-HRP. c, ELISA of hu1B7 on a 4 nm coat of PTx or S1-220K, detected by GαhFc-HRP.

FIGURE 4.

PTxS1 conformational epitope determination. a, a subset (29/220 residues) of the fitness-metric heat map for the PTxS1-hu1B7 interaction. Sequence entropy for the unselected/display population (green) and unselected/bound population (black) is plotted below with their respective cut-offs (dashed lines). b, subtractive sequence entropy analysis for PTxS1-hu1B7interaction. The light gray surface represents the S1 subunit, and the dark gray represents other subunits of PTx. Conserved residues (orange) are found on the S1 subunit proximal to the S5 and S6 subunits. Non-conserved residues (purple) are found over most of the solvent-accessible surface area. c, close-up view of the conserved residues at the epitope interface. PTxS1 is represented with a schematic and sticks format, whereas the other subunits are represented as the dark gray surface.

FIGURE 5.

Fitness metric and relative dissociation constant error. a, relative dissociation constant as a function of fitness metrics. The vertical dashed line represents the average fitness metric for stop codon positions. b, standard error as a function of fitness metric for different numbers of unselected counts (red, 10; blue, 20; green, 30; orange, 50; purple, 100; black, 500). Fitness metrics associated with lower number of counts have higher error. c, relative dissociation constant error as a function of relative dissociation constant for different numbers of unselected counts. As the relative dissociation constant increases the amount of error increases.

FIGURE 6.

TROP2 expression in MDA-MB-231 cells enhances migration and invasion. a and b, MDA-MB-231 cells were transfected with scramble siRNA or siRNA targeting TROP2 for 24 h. The mRNA (a) and protein levels (b) of TROP2 were measured by real-time PCR and Western blotting. n = 3. **, p < 0.01; ***, p < 0.001 versus control. c, suppression of MDA-MB-231 migration by silencing TROP2. MDA-MB-231 cells were transfected with scramble siRNA or siRNA targeting TROP2 and then subjected to a transwell migration assay. The cells were allowed to migrate toward serum for 8 h. Triplicate wells were used for each condition in three independent experiments. ***, p < 0.001 versus control. d, silencing TROP2 reduced the migration of highly invasive breast cancer cells. MDA-MB-231 cells were transfected with scramble siRNA or TROP2 siRNA and subjected to a wound-healing assay. Representative photographs at the indicated time points from three independent experiments, with each performed in triplicate wells. Magnification, 10×. e, suppression of MDA-MB-231 invasion by silencing TROP2. MDA-MB-231 cells were transfected with scramble siRNA or siRNA targeting TROP2 and then seeded in a Matrigel-coated Boyden chamber and subjected to a transwell invasion assay. The cells were allowed to migrate toward serum for 20 h. Triplicate wells were used for each condition in three independent experiments. ***, p < 0.001 versus control.

FIGURE 7.

TROP2 conformational epitope determination. a, the domains of membrane protein TROP2. The extracellular portion of TROP2 (TROP2Ex) contains an N-terminal domain (ND, green), TY domain (brick red), and C-terminal domain (CD, cyan). TM indicates the membrane spanning portion, and Trop2Ic is the intracellular domain. b, homology model of TROP2Ex homodimer shown in surface view. One subunit is colored by sequence entropy based on m7e6 binding (orange, conserved; purple, non-conserved; gray, intermediate). The other subunit is colored by domain using the same coloring scheme as in panel a. The fine epitope is located on the membrane distal face of the C-terminal domain. The center of the epitope on one subunit is separated by 4.1 nm from the epitope on the adjacent subunit. c, bar graphs showing the number of migrating cells in MDA-MB-231 cells treated with PBS (control) or m7EG IgG. d, representative bright field images of Boyden's chamber inserts showing cells that migrated across the 8-μm membrane after 24 h of treatment. e, bar graphs showing number migrating cells with control or m7E6 Fab treatment. f, representative bright field images showing migrating cells across the membrane. p values indicate significance of difference in mean (n = 3) determined using Student's two-tailed t tests.

FIGURE 8.

a, 10⁵ MDA-MB-231 cells were labeled with 50 nm biotinylated m7e6 IgG (orange), 50 nm biotinylated m7e6 Fab (cyan), or nude (blue) in buffer PBS plus 1 g/L bovine serum albumin for 30 min at room temperature. After washing cells were secondarily labeled with streptavidin-phycoerythrin and processed by flow cytometry. b, bar graphs indicating the average (n = 3) Alamar Blue assay absorbance (570 nm) in cells treated with PBS (control) or increasing concentration of m7E6 IgG (10–40 μg/ml). c, bar graphs indicating the average (n = 3) lactate dehydrogenase absorbance (590 nm) in control and m7EG IgG-treated cells. p values indicate the significance levels of the influence of increasing concentrations of the IgG treatment on proliferation or cytotoxicity levels determined using one-way analysis of variance.

FIGURE 9.

Confocal images showing expression and localization of TROP2 intracellular domain represented by green fluorescence in MDA-MB-231 cells treated with PBS (control), m7E6 IgG, or Fab with nuclear counter-staining indicated by blue fluorescence. The individual panels were recorded at 60× magnification (scale bar = 50 μm) with identical image acquisition parameters between different conditions.