Comprehensive elucidation of the structural and functional roles of engineered disulfide bonds in antibody Fc fragment

Abstract

Therapeutic monoclonal antibodies and Fc-fusion proteins containing antibody Fc fragment may tend to destabilize (e.g. unfold and aggregate), which leads to loss of functions and increase of adverse risks. Although engineering of an additional disulfide bond has been performed in Fc or Fc domains for optimization, the relationships between introduced disulfide bond and alteration of the stability, aggregation propensity and function were still unclear and should be addressed for achievement of better therapeutic outcome. Here, we constructed three human IgG1 Fc mutants including Fc^CH2-s-s- (one engineered disulfide bond in CH2 domain), Fc_CH3-s-s- (one engineered disulfide bond in CH3 domain), and Fc_CH3-s-s-^CH2-s-s- (two engineered disulfide bonds in CH2 and CH3 domains, respectively) for evaluation. As expected, each mutated domain shows obviously increased stability during thermo-induced unfolding, and Fc_CH3-s-s-^CH2-s-s- is most thermo-stable among wildtype Fc (wtFc) and three mutants. The order of overall stability against denaturant is Fc_CH3-s-s-^CH2-s-s- > Fc^CH2-s-s- > Fc_CH3-s-s- > wtFc. Then the aggregation propensity was compared among these four proteins. Under conditions of incubation at 60 °C, their aggregation resistance is in the order of Fc_CH3-s-s-^CH2-s-s- > Fc^CH2-s-s- > Fc_CH3-s-s- ≈ wtFc. In contrast, the order is Fc_CH3-s-s-^CH2-s-s- > Fc_CH3-s-s- > Fc^CH2-s-s- ≈ wtFc under acidic conditions. In addition, the Fc-mediated functions are not obviously affected by engineered disulfide bond. Our results give a comprehensive elucidation of structural and functional effects caused by additional disulfide bonds in the Fc fragment, which is important for Fc engineering toward the desired clinical performance.

Keywords: Fc engineering; Fc fragment; Fc receptor; aggregation; circular dichroism (CD); disulfide bond; function; fusion protein; monoclonal antibody; stability.

Figure 1.

Design of Fc mutants with additional disulfide bond. A, the structure of wtFc obtained from an intact IgG1 with solved crystal structure (Protein Data Bank code 1HZH (12)) presented by PyMOL (PyMOL Molecular Graphics System, version 1.2r 1 Schrödinger, LLC). The CH2 and CH3 domains are colored cyan and green, respectively. The native cysteines that form disulfide are colored yellow. The distances of two α-carbon atoms in CH2 and CH3 are 6.6 and 6.5 Å, respectively. Asn²⁹⁷ colored purple is the glycosylation site. Residues Leu²⁴² and Lys³³⁴ colored red in CH2 could be mutated to two cysteines to introduce the additional disulfide bond (the distance of two α-carbon atoms is 6.7 Å), while residues Pro³⁴³ and Ala⁴³¹ colored orange in CH3 could be replaced by two cysteines to get the engineered disulfide bond (the distance of two α-carbon atoms is 5.7 Å). B, sequence alignment of wtFc and designed mutants Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s-.

Figure 2.

Molecular mass of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s-. A, SDS-PAGE of purified wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and FcCH2-s-s CH3-s-s-. B, SEC analysis of purified wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s-. The standard curve is used for calculation of molecular mass.

Figure 3.

Thermostability of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s-. A, secondary structure at 25 °C measured by CD. A major negative peak at 216 nm in each protein indicates typical β-sheet structure. B, thermo-induced unfolding curve measured by CD. The CD signal was recorded at 216 nm in the temperature range from 25–94 °C with heating rate of 0.5 °C/min. Experiments were repeated at least twice, and the results from one representative experiment are presented.

Figure 4.

Urea-induced unfolding of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s-. The fraction folded (ff) of the protein was calculated as ff = ([θ] − [θ_U])/([θ_F] − [θ_U]). [θ_F] and [θ_U] were the mean residue ellipticities at 216 nm of folded state (F) in the absence of urea and unfolded state (U) in the presence of urea concentration of 10 m. The urea concentrations at half of unfolding course of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s- are 7.3, 8.7, 7.8, and 9.0 m, respectively. Experiments were repeated at least twice, and the results from one representative experiment are presented.

Figure 5.

Aggregation resistance of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s- measured by turbidity assay and size-exclusion chromatography. A, turbidity assay of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s- after 60 °C incubation at different time points. B, evaluation of aggregation formation by SEC. In addition to Peak 1, there is an obvious aggregation peak (Peak 2) in wtFc and Fc_CH3-s-s-, respectively, at the end of 60 °C incubation. Fc_CH3-s-s-^CH2-s-s- and Fc^CH2-s-s- are more resistant to aggregation. However, compared with Fc_CH3-s-s-^CH2-s-s-, a very small Peak 2 could be observed in Fc^CH2-s-s-. The inset is a standard curve as used above. Experiments were repeated twice, and the results from one representative experiment are presented.

Figure 6.

The aggregation tendency of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s- in acidic conditions. Sample aliquots were taken at predetermined time intervals and analyzed by recording the absorbance at 320 nm. Experiments were repeated twice, and the results from one representative experiment are presented.

Figure 7.

Flow cytometry for measurement of binding of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s- to FcγRI. A, FcγRI-positive U937 cells were incubated with 40 nm wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s- proteins. The obvious fluorescence intensity shift could be observed. In the case of FcγRI-negative HeLa cells, the shift is minor. B, fluorescence intensity shifts at different protein concentrations from 0 to 1,000 nm. All four proteins bind to U937 cells in a dose-dependent manner. MFI, mean fluorescence intensity.

Figure 8.

Determination of binding of wtFc, Fc^CH2-s-s-, Fc_CH3-s-s-, and Fc_CH3-s-s-^CH2-s-s- to shFcRn at pH 6.0 by BLI. In general, all four proteins bind to shFcRn with high similarity. The experiments were repeated twice, and the results from one representative experiment are presented.