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

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 FcCH2-s-s- (one engineered disulfide bond in CH2 domain), FcCH3-s-s- (one engineered disulfide bond in CH3 domain), and FcCH3-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 FcCH3-s-s-CH2-s-s- is most thermo-stable among wildtype Fc (wtFc) and three mutants. The order of overall stability against denaturant is FcCH3-s-s-CH2-s-s- > FcCH2-s-s- > FcCH3-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 FcCH3-s-s-CH2-s-s- > FcCH2-s-s- > FcCH3-s-s- ≈ wtFc. In contrast, the order is FcCH3-s-s-CH2-s-s- > FcCH3-s-s- > FcCH2-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.

The Fc-based therapeutic proteins include therapeutic monoclonal antibodies and Fc-fusion proteins. As their common part, antibody Fc fragment is important for maintenance of the molecular structure and function (1). Because instability and aggregation tendency constrain the development of these therapeutic proteins for clinical use, engineering of Fc fragment for improvement of the physicochemical properties including stability and aggregation resistance could be a useful platform for overcoming the obstacles. Many methods, including introduction of covalent bond (e.g. disulfide bond) and modification of noncovalent bond, have been used for optimization of Fc fragment to achieve the expected clinical outcomes (1).
The Fc fragment in an Ig is dimeric form composed of two copies of CH2 domains and two copies of CH3 domains. In each domain, there is a native disulfide bond that is important for the structural stability (2). It has been shown that the native disulfide bond between Cys 367 and Cys 425 (all the Fc residues here are numbered according to EU numbering (2)) can support the folding of single CH3 domain, as well as the dimerization process between two CH3 domains, and prevent aggregation formation (3)(4)(5). Although the role of native disulfide between Cys 261 and Cys 321 in the CH2 domain has not been well characterized, we could not observe the soluble expression of isolated CH2 in Escherichia coli after mutations of two native cysteines to other residues (data not shown). Hence, the native disulfide bond should also play an important role in supporting the correct folding of CH2 domain. Because of the important roles of native disulfide bond, it could be highly desired that introduction of additional disulfide bond could stabilize the Fc molecule to make it better toward clinical use.
In a previous study, we engineered an additional disulfide bond by replacement of Phe 242 and Lys 334 by two cysteines in isolated wildtype CH2 domains (wtCH2) from the IgG1 Fc fragment to identify a mutant termed m01 (6). The melting temperature (T m ) of m01 is ϳ18°C higher than that of wtCH2, while the urea concentration of 50% unfolding rises from 4.2 to 6.8 M. No obvious conformational change occurs. Following this study, we and other group also reported that introduction of an additional intradomain disulfide bond by mutation of certain amino acids (e.g. replacement of Pro 343 and Ala 431 by two cysteines, respectively) in CH3 domain leads to ϳ8°C or ϳ35°C increases of the T m in the context of an intact Fc fragment or an isolated monomeric CH3 domain, respectively (7,8). In addition, introduction of interdomain disulfide by replacement of a C-terminal Lys 447 to cysteine and mutation of other nearby residues in CH3 domain can also increase the stability and aggregation resistance of the cyclized Fc fragment (9, 10). In a recent study, more residues in CH2 domain were selected for induction of additional disulfide bonds, which results in various effects on stability and circulation in vivo (11).
Although current results show that engineering of disulfide bond in Fc is promising for modification of therapeutic monoclonal antibodies and Fc-fusion proteins, it is still not very clear how these introduced disulfide bonds in different domains of Fc fragment contribute to optimization on structural and functional properties of Fc. For example, because unpaired cysteines may mismatch and form big oligomer, more disulfide bonds may increase the risk of aggregation. Therefore, the potential change of aggregation propensity of Fc fragment with extra disulfide bonds should be also well evaluated.
To address these issues, we constructed three Fc mutants with additional disulfide bonds in the CH2 domain, the CH3 domain, and both the CH2 and CH3 domains and expressed them in mammalian cells. After purification, a series of experiments were performed for evaluation of the influence of engineered disulfide bonds on structural and functional properties. We found that introduction of disulfide bonds in different domains could make different contributions on improvement of physiochemical properties including stability and aggregation resistance. One Fc mutant with two extra disulfide bonds in the CH2 and CH3 domains, respectively (Fc CH3-s-s-CH2-s-s-) has the best physicochemical properties without obvious alteration on Fc-mediated functions. Our results give straightforward evidence that Fc-based therapeutics could be improved through engineering of disulfide bonds in the Fc fragment.

Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-sexist as dimeric form
wtFc and its three mutants could be solubly expressed at comparable level in mammalian cells and purified on protein G resin. Because their theoretic molecular mass calculated by amino acid residues was ϳ24 kDa, and all of them should be glycosylated at Asn 297 , the size of corresponding bands was correct as shown in SDS-PAGE ( Fig. 2A). To determine the molecular mass of wtFc and its mutants in solution, all four proteins were analyzed with size-exclusion chromatography (SEC) (Fig. 2B). They showed identical elution profiles that only had one unique peak with molecular mass of ϳ60 kDa calculated by molecular mass standards (Fig. 2B), which indicated that all four proteins existed in a dimeric form. Therefore, the cysteines could be paired correctly to form intradomain disulfide bond, and the dimerization of each monomeric Fc fragment was not affected as desired.

Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-sare significantly more stable than wtFc
The secondary structure of these four proteins were measured by CD. The spectra showed that their structure is very similar, consisting of primarily of ␤-strands at 25°C. Hence, introduction of disulfide bond did not change the overall structure (Fig. 3A). Then the thermal stability of wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swas evaluated (Fig. 3B). The unfolding curves of Fc and its mutants included unfolding of the CH2 and CH3 domains. In the case of wtFc and Fc CH3-s-s-, two separate courses, representing unfolding processes of CH2 and CH3 domains, could be obviously observed. The T m values of CH2 domains in wtFc and Fc CH3-s-s-were 73.3°C, while the T m of CH3 domain in wtFc was 84.3°C. Notably, the T m of CH3 domain in Fc CH3-s-s-dramatically increased because of the engineered disulfide bond, which could not be accurately calculated because the unfolding was not completed at 94°C. In contrast to that of wtCH2 and Fc CH3-s-s-, the unfolding course was uniform in the cases of Fc CH2-s-sand Fc CH3-s-s-CH2-s-sas observed because of the increase of thermoresistance in CH2 domain after introduction of an additional disulfide bond. Therefore, the total T m of Fc CH2-s-scould be obtained, which was 85.2°C. Notably, the total T m of Fc CH3-s-s-CH2-s-scould be very high (e.g. Ͼ 90°C). Because separate introduction of disulfide bonds in CH2 and CH3 domains obviously increased the T m of each engineered domains, simultaneous introduction of two disulfides in two domains resulted in extreme thermostability of Fc CH3-s-s- The stability against chemically induced unfolding of wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swas also compared in the presence of urea (Fig. 4). The 50% unfolding of wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-soccurred at urea concentrations of 7.3, 8.7, 7.8, and 9.0 M, respectively. Therefore, introduction of disulfide bond in CH2 domain could increase the stability against chemical denaturant more obviously than that in CH3 domain. In general, the trend of improvement of ability against unfolding under thermal pressure and in the presence of denaturant after engineering disulfide bond was very similar, although the contributions from different disulfide bonds may vary.

Additional disulfide bond can increase aggregation resistance
Turbidity assay was performed for evaluation of aggregation propensity under high temperature and acidic conditions. The rates of increase of the absorbance at 320 nm in wtFc and Fc CH3-s-swere faster than that in Fc CH2-s-sand Fc CH3-s-s-CH2-s-safter incubation at 60°C, which indicated more aggregates formed in wtFc and Fc CH3-s-s-under high temperature pressure (Fig. 5A). The result was confirmed by size-exclusion chromatography at the end of incubation, which showed that no obviously/or very 3 minor soluble big oligomers formed in the case of Fc CH3-s-s-CH2-s-sand Fc CH2-s-s- (Fig. 5B). In contrast, a remarkable second peak eluted earlier in wtFc and Fc CH3-s-s-was observed indicating formation of aggregates. Under acidic conditions, Fc CH3-s-s-CH2-s-sand Fc CH3-s-s-were more aggregation-resistant than Fc CH2-s-sand wtFc (Fig. 6). Although the introduced disulfide bond in CH2 domain did not significantly change the aggregation propensity of Fc CH2-s-sat low pH compared with wtFc, it could cooperate with the additional disulfide bond in CH3 domain to further enhance the aggregation resistance (Fc CH3-s-s-CH2-s-sversus Fc CH3-s-s-). Hence, engineering of disulfide bonds in Fc can improve its aggregation resistance. The different positions of introduced disulfide bonds could have different contributions for increase of aggregation resistance under different conditions.

CH2-s-sto Fc receptor
Although the positions of mutated cysteines were not involved in Fc␥ receptor (Fc␥R) binding (13), it might affect the interaction by conformational change. Therefore, U937, a high Fc␥RI-expression cell line, was used here for measurement of binding of Fc and its mutants to Fc␥RI, which could be a representative for estimation of binding to Fc receptors (14). The obvious fluorescence intensity shift was observed when wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swere added into U937 cells, indicating their binding to Fc␥RI (Fig. 7). All four proteins exhibited very similar binding behaviors, which meant that additional disulfide bond did not affect the interaction with effector molecules and subsequent Fc-mediated effector functions.  (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 ␣-carbon atoms in CH2 and CH3 are 6.6 and 6.5 Å, respectively. Asn 297 colored purple is the glycosylation site. Residues Leu 242 and Lys 334 colored red in CH2 could be mutated to two cysteines to introduce the additional disulfide bond (the distance of two ␣-carbon atoms is 6.7 Å), while residues Pro 343 and Ala 431 colored orange in CH3 could be replaced by two cysteines to get the engineered disulfide bond (the distance of two ␣-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-.

Binding of wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-sto neonatal Fc receptor (FcRn) at pH 6.0
Because binding of antibody Fc fragment to FcRn in a pH-dependent manner is one of the key factors for extension of the serum half-life of antibody in vivo (15), the interaction of these four proteins with soluble human FcRn (shFcRn) was analyzed by biolayer interferometry technology (BLI) for evaluation of their serum half-lives after introduction of disulfide bond (Fig.  8). As measured, all the Fc and its mutants bound to FcRn at pH 6.0 with relatively low affinity in range of submicromole to micromole ( Table 1). The response curve completely dropped to baseline level when the probe was immersed into pH 7.4 buffer, which indicated FcRn-bound proteins were completely released (data not shown). Although the measured affinities of these four proteins to FcRn had some difference, possible because of different structural stability at acidic conditions, the overall binding pattern of them to FcRn was still quite similar and the introduced disulfide bond might not reduce the serum half-life in vivo.

Discussion
Optimization of Fc fragment is desired to achieve better clinical potentials, which includes modification of physiological functions and improvement of structural properties. To modify Fc functions (e.g. enhancement of FcRn binding and modulation of effector functions), several studies focused on engineering CH2 domain. However, these modifications might decrease the stability of the whole Ig molecule (11,16,17), which limits the further application for clinical use.
We and another group previously disclosed that CH2 domain in Fc fragment is relatively unstable against heating and chemical reagent-induced unfolding compared with CH3 domain (6,8,18). Interestingly, it has also been shown that Fc unfolding is first triggered by the protonation of acidic residues on CH2 domain under acidic conditions (19). Therefore, CH2 domain is an ideal target for improvement of the stability and aggregation resistance of Fc. We previously identified a CH2 mutant (m01s) that has increased stability, aggregation resis-

Engineering of antibody Fc fragment with disulfide bond
tance, and pH-dependent binding to FcRn by introducing an additional disulfide bond and removal of several unstructured residues at the N terminus (6,20,21). A recent work found that introduction of a disulfide bond could compensate the reduction of stability caused by aglycosylated IgG (11). In addition to introduction of a disulfide bond, site-directed mutagenesis at several hot spots in the CH2 domain could also increase the stability and aggregation resistance in the context of an intact IgG by modification of noncovalent interactions (22). In addition, replacement of an enhanced aromatic sequon into the N-glycosylated loop DE has been performed that could enhance its thermal stability and resistance of aggregation induced by low pH in the context of a full-length IgG1 (23). The strong interactions between two CH3 domains are important for Fc dimerization. Therefore, the physicochemical properties of CH3 domain also have significant effects on the structural stability of Fc. In a previous study, the stability of bovine IgG1 CH3 could be increased by mutation of several residues according to its human partner (24). As mentioned above, the disulfide bond could be introduced into different positions in CH3 domains to enhance the stability of intact Fc fragment and isolated monomeric CH3 domain. Therefore, introduction of additional disulfide bond could be an attractive direction to modify Fc fragment.
According to our results, introduction of disulfide bond in CH2 domain (Fc CH2-s-s-) results in ϳ10°C increase in T m of CH2 domain in the context of Fc fragment, as well as 1.4 M increase in the concentration of urea at 50% unfolding of Fc fragment. In contrast, additional disulfide bond in CH3 domain (Fc CH3-s-s-) also leads to an ϳ10°C increase in T m of CH3 domain but only a 0.5 M increase in the urea concentration at middle point of unfolding. The Fc mutant with two additional disulfide bonds (Fc CH3-s-s-CH2-s-s-) has extremely high T m (Ͼ90°C) and very strong resistance to urea-induced unfolding (concentration at middle point of unfolding is 9.0 M). Based on thermo-induced unfolding experiment, the separate introduction of disulfide bonds into CH2 or CH3 domains could only increase the stability of the corresponding domain because the folding of them is independent. Interestingly, it is observed that the T m value of CH2 in Fc CH3-s-s-CH2-s-sis higher than that in Fc CH2-s-s- (Fig.  3), which may be caused by synergic effect derived from simultaneously engineering disulfide bonds in CH2 and CH3 domains.
In the evaluation of aggregation formation under 60°C incubation, introduction of disulfide bond in CH2 domain can prevent aggregation much better than that in CH3 domain. Because the thermostability of CH2 is much lower than that of CH3, the thermo-induced aggregations mainly depend on aggregation and unfolding tendency of CH2. Therefore, stabilization of CH2 domain is the major factor to determine the aggregation resistance of a whole Fc molecule under heating conditions.
In acidic conditions, it has been shown that CH3 plays critical role in driving intact antibody aggregation among antibody  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-sand Fc CH2-s-sare 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.

Engineering of antibody Fc fragment with disulfide bond
domains (25). In accordance with this result, we also found that additional disulfide in CH3 domain (Fc CH3-s-s-) could make Fc more aggregation-resistant than that in the CH2 domain (Fc CH2-s-s-) under pH 2.0. Therefore, stabilization of CH3 could increase the aggregation resistance of the Fc fragment to low pH more significantly than that of CH2. Notably, although it seems that only engineered disulfide bond in CH2 domain has no obvious contribution to improvement of aggregation resistance to low pH, the synergic effect is still very obvious (Fig. 6). Hence, the effect on stabilization of wtFc could be maximized when CH2 and CH3 domains are engineered with additional disulfide bonds together.
Because introduction of disulfide bond may cause the loss of Fc-mediated functions, we also compared the function of these Fc mutants. All the Fc mutants with additional disulfide bonds still maintain their Fc receptor-binding abilities as wtFc. It has also been reported that the introduced disulfide bond did not obviously change the binding to FcRn (7,11). Although we observed some slight difference of the affinity to FcRn after engineering of disulfide bond, the general binding was still in a pH-dependent manner. Therefore, the recycling of the protein might not significantly change (e.g. reduce).
In summary, we demonstrated the contribution of introduced disulfide bonds in different Fc domains to the stability, aggregation resistance, and function. The most stable and aggregation-resistant mutant Fc CH3-s-s-CH2-s-snewly identified here could be used for modification of Fc-based therapeutics toward better clinical outcomes.

Thermal stability measurement by CD
The secondary structures of wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swere determined by CD spectroscopy. The purified

Engineering of antibody Fc fragment with disulfide bond
proteins were dissolved in PBS (pH 7.4) at the final concentration of 0.4 mg/ml, and the CD spectra were recorded on an Applied Photophysics Chirascan-SF.3 spectrophotometer (Applied Photophysics Ltd.). Wavelength spectra were recorded at 25°C using a 0.1-cm-path length cuvette for native structure measurements. Thermal stability was measured by recording the CD signal at 216 nm in the temperature range from 25 to 94°C with heating rate of 0.5°C/min. The experiments were repeated twice, and the T m values were presented as means Ϯ S.D.

Spectrofluorometry
The purified wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swere added into PBS (pH 7.4) with urea from 0 to 10 M. The final concentration of each protein was 100 g/ml. After incubation at 25°C overnight, the intrinsic fluorescence intensity was recorded on EnVision TM (PerkinElmer) to compare their stability against chemical denaturant. The measurement was performed with excitation wavelength at 280 nm and emission spectra at 340 nm at 25°C as previously reported (6). The background fluorescence intensity of the solution (buffer ϩ denaturant) was deducted from the sample fluorescence intensity. The experiments were repeated twice, and the values of urea concentration at 50% unfolding of each proteins were presented as means Ϯ S.D.

Turbidity assay
The purified wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swith concentration of 2 mg/ml were prepared by filtering through 0.22-m filter and centrifuging at 13,000 ϫ g for 10 min to make a final 900 l of working solution. After incubation at 60°C,turbiditywasquantifiedbyrecordingthe320-nmabsorbance at different time points with UV spectrophotometer (Beijing Liuyi Biotechnology Co., Ltd.). After incubation at 60°C for 552 h (23 days), the protein samples were centrifuged at 4°C, 13,000 ϫ g for 15 min, and then the supernatant was injected onto a Superdex 75 10/300 GL column running on ÄKTA pure system to assess oligomer formation. To evaluate their behaviors in acid-induced aggregation, the final concentration of each protein was also adjusted to 2 mg/ml in PBS (pH 2.0 adjusted by HCl). The samples were

Engineering of antibody Fc fragment with disulfide bond
incubated for up to 34 days at 37°C without agitation. Turbidity was measured by recording the 320-nm absorbance at different time points.

Flow cytometry
To measure the interactions of wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swith U937 cells, which can highly express Fc␥RI (14), each of these four proteins with different concentrations in PBS containing 1% BSA (w/v) were incubated with U937 cells at 4°C for 1 h. Unbound proteins were washed away with PBS. The goat anti-human IgG Fc cross-adsorbed antibody, DyLight 650 (Invitrogen), used as secondary antibody, was incubated with cells at 4°C for 1 h. The cells were washed and resuspended in PBS (pH 7.4) for measurement of the fluorescence intensity on CytoFLEX (Beckman Coulter). HeLa cells were used as negative control.

Design, expression, and purification of shFcRn
The genes encoding the ␣-chain and ␤2-microglobulin of shFcRn were cloned into the dual promoter vector pVITRO2neo-mcs (Invivogen) to construct the recombinant expression plasmid pVITRO2-shFcRn. This plasmid was transfected into 293F cells for expression as described above. After 5 days of expression, shFcRn heterodimer was purified from supernatant using a human IgG conjugated Sepharose column (provided by Professor Bing Yan). The shFcRn was bound in PBS at pH 6.0, followed by elution in PBS at pH 7.4. The concentration was determined by NanoPhotometer N60 with extinction coefficient 1.78.

Binding of wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-sto shFcRn
Real-time binding assay between protein FcRn and wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-swas carried out on an Octet QK system (Fortebio) at 25°C using BLI. Specifically, the shFcRn was labeled with EZ-Link sulfo-NHS-LC-biotin (Pierce) as descried in its instructions. After labeling, the shFcRn was desalted to remove nonreacted biotin with Amicon Ultra-0.5 centrifugal filter unit, 3 kDa (Merck). Then the biotinlabeled shFcRn was loaded into streptavidin biosensors, and wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-sdissolved in PBS containing 0.02% Tween 20 (pH 6.0) were added at a series of concentrations: 5000, 1667, 556, and 185 nM. The affinities of wtFc, Fc CH2-s-s-, Fc CH3-s-s-, and Fc CH3-s-s-CH2-s-sto FcRn were calculated according to 1:1 binding model through the Octet QK software package (Fortebio), and the equilibrium dissociation constant (K D ) value was equal to the kinetic dissociation rate constant divided by the kinetic association rate constant. After the dissociation step, the regeneration step was performed by changing biosensors into pH 7.4 buffer to make all bound proteins completely dissociate from the biosensors and the baseline return to initial state.