Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Abstract

Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTM-targeting antibodies have reported frequent nonspecific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phosphothreonine 231 (pThr-231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinity-matured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved >20-fold over that of the WT antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity toward the nonphosphorylated target site but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phosphotau antibody. In cell- and tissue-imaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTM-specific antibodies and highlight the importance of assessing the specificity in the antibody engineering process.

Keywords: affinity; anti-PTM antibody; antibody; antibody specificity; immunochemistry; neurodegeneration; phospho-specific; post-translational modification; protein phosphorylation; tau protein (tau).

Figure 1.

Schematic representation of antibody-specificity quantification using yeast surface display. Individual peptide epitope binding to single-chain variable region fragments displayed on yeast can be quantified. By labeling multiple peptides with distinct tags, competitive binding of potentially cross-reacting epitopes, such as a phosphopeptide and nonphosphopeptide (peptide mixture indicated by a dotted box) can be quantified using multicolor flow cytometry.

Figure 2.

Characterization of yeast-displayed WT pThr-231 scFv. a, confocal microscope images of yeast-displayed WT pThr-231 scFv and an isotype-matched control scFv. scFv expression shown in red was detected using a mouse anti-FLAG antibody to assess full-length scFv expression. Cognate Pphos binding is indicated in green. Scale bars, 5 μm. b, affinity measurement of yeast-displayed WT pThr-231 scFv. The yeast cells were incubated with a serial dilution of Pphos. Error bars indicate S.D. from three independent experiments. c, assessment of WT pThr-231 scFv specificity. Flow cytometry dot plots for yeast-displayed WT pThr-231 scFv (top row) or an isotype-matched control scFv (bottom row). The first column shows data for scFv expression and Pphos-b binding. In the following columns, yeast cells were incubated with a mixture of either Pnonphos-b and Pphos-A488 (second column), Pscram-b and Pphos-A488, or Pphos-b and Pphos-A488, all at 0.5 μm.

Figure 3.

Directed evolution of yeast-displayed pThr-231 scFv variants with improved affinity. a, flow chart for the directed evolution process. High-affinity variants were sorted using FACS. b, flow cytometry dot plot with a sorting gate used for screening cells with scFv expression and high Pphos-b binding. c, dot plots comparing yeast displaying WT pThr-231 scFv (left) and a yeast pool resulting from three rounds of sorting and mutagenesis (right) were labeled for scFv expression and Pphos-A488 binding.

Figure 4.

Amino acid sequence of individual mutants isolated from the screen for improved affinity. The sequences in the boxes indicate CDRs. High-occurrence mutations are indicated in bold. Kabat numbering is indicated at the top.

Figure 5.

High-occurrence mutations identified from the screen mapped on the WT pThr-231 structure. Heavy chain is shown in green, and light chain is shown in purple. Positions of high-occurrence mutations are indicated in red and labeled with the WT amino acid. Boxed amino acids indicate those mutated in scFv 3.24. The structure was generated using CCP4MG, based on Protein Data Bank code 4GLR.

Figure 6.

Characterization of affinity and specificity of individual scFvs. a, binding intensity normalized to the expression level of yeast-displayed WT pThr-231 scFv and mutants to Pphos-b at 2 nm. The rightmost sample is the WT pThr-231 scFv incubated with Pphos-b at 1 μm as a positive control. b, affinity titration for yeast-displayed scFv 3.05. c and d, binding intensity of yeast-displayed WT pThr-231 scFv and mutants to Pnonphos-b at 1 μm (c) and Pscram-b at 1 μm (d), respectively. e, affinity titration for yeast-displayed scFv 3.24. f, binding intensity normalized to the expression level of yeast-displayed WT pThr-231 scFv and mutants to Pnonphos-b at 1 μm. Error bars indicate S.D. from three independent experiments. Statistics for a, c, d, and f, Dunnett's multiple comparisons test; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; otherwise, p > 0.05.

Figure 7.

Cell labeling using purified pThr-231 scFvs. a, confocal microscope images of purified, fluorescently labeled WT pThr-231 scFv and mutant 3.24 binding to HEK293FT cells expressing WT tau (top row), WT tau treated with λ-phosphatase (Lambda PP) (second row), tau T231A (third row), and tau T231E (bottom row). All human tau constructs were fused to EGFP at the N terminus (EGFP-tau). The first column shows DAPI staining in blue. The second column shows tau expression in green. The third column shows scFv binding in red. Scale bars, 50 μm. b, quantification of the fluorescence intensity of WT pThr-231 scFv and mutant 3.24 binding to HEK293FT cells expressing WT tau at scFv concentrations from 16 pm to 50 nm. Error bars indicate S.D. from three independent experiments. Statistics for b, Student's t test; *, p ≤ 0.05. MAPT, microtubule-associated protein tau.

Figure 8.

Tissue labeling using purified pThr-231 scFvs. a, immunohistochemistry using the scFvs on brain slices of 8-month-old mice expressing P301S mutant tau indicates that both scFvs robustly identify neurofibrillary tangles in tissue. Each scFv was used at 50 and 10 nm to identify optimal staining concentrations and show a dose response for staining intensity, quantified in b. Tau is shown in red with nuclei stained using DAPI in blue. The 10 nm concentrations showed significant reduction in staining intensity for both the WT and 3.24 scFv (****, p < 0.0001 by one-way analysis of variance with p < 0.0001 between the 50 and 10 nm working concentrations of each scFv using Tukey's multiple comparisons test; error bars indicate S.D. of all data points). There were no statistical differences between the WT and 3.24 scFv at either concentration. c, neither scFv stained WT or tau knockout mouse tissue at the 50 nm working concentration, indicating no off-target staining or background fluorescence. d, immunohistochemistry of human AD and aged control tissues also indicates that both scFvs stain phosphotau tangles in disease (scFv concentration, 50 nm). However, in the human tissue, the high-affinity scFv 3.24 shows a significant increase in staining intensity compared with the WT scFv, which is quantified in e (****, p < 0.0001 using a two-sample t test assuming unequal variances; error bars indicate S.D. of all data points).