Abstract
Incorporation of noncanonical disulfide linkages into single‐domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here, we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures (T ms), reversibility of thermal unfolding, solubility, and antigen‐binding affinities of three types of sdAbs (VHH, VH, and VL domains). The Cys40‐Cys55 disulfide linkage was tolerated by 9/9 VHHs, 12/12 VHs, and 2/11 VLs tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40‐Cys55 disulfide linkage increased sdAb T m by an average of 10.0°C (range: 0–21.8°C). However, enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as, for some sdAbs, significantly decreased solubility and antigen‐binding affinity. Thus, Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains, although its effects should be tested empirically for individual sdAbs.
Keywords: single‐domain antibody, disulfide linkage, thermostability, protein engineering
Short abstract
PDB Code(s): 4NBZ
Introduction
Disulfide linkages play a critical role in the folding and stability of secreted proteins. For protein therapeutics, thermostability is a critical parameter affecting manufacturability,1 safety,2 and efficacy.3, 4 Starting in the mid‐1980s, exogenous disulfide linkages have been introduced into a wide variety of proteins, resulting in engineered variants with improved thermodynamic and kinetic stability.5
Antibodies (IgGs) are large heterodimeric macromolecules bearing multiple intra‐ and interchain disulfide linkages. Despite the complexity of these molecules, introduction of noncanonical disulfide linkages into antibody constant regions (CH2 and CH3 domains) improved the thermostability both of the individual domains and of the full‐length IgG.6, 7, 8 Disulfide engineering of antibody variable domains carries the additional challenge of improving stability without compromising antigen‐binding affinity.9 However, in addition to the conserved Cys23‐Cys104 linkage (IMGT numbering used throughout) present in nearly all antibody variable domains, noncanonical disulfide linkages frequently occur naturally within the variable domains of some species' antibodies (e.g., camelids, sharks, and cows) and thus are clearly compatible with antigen recognition.
Single‐domain antibodies (sdAbs), the variable domains of camelid and shark heavy chain‐only antibodies, are typically highly thermostable in comparison with other types of antibody fragments. However, additional stability may be of value in some applications (e.g., oral or inhaled administration), especially for synthetic sdAbs, some of which tend to have lower melting temperatures (T ms) than naturally occurring sdAbs.10 In the mid‐2000s, two groups independently identified a rare naturally occurring noncanonical disulfide linkage formed between Cys54 and Cys78 of a camelid sdAb, and showed that introducing this linkage improved the thermostability of other sdAbs while sparing antigen binding.11, 12 This work was later replicated13, 14, 15, 16, 17, 18, 19, 20 and extended to synthetic human autonomous VH 21 and VL 22 domains as well as the VH domains of Fabs8 and full‐length IgGs.23 Saerens et al. also showed that thermostability of camelid sdAbs could be improved to a lesser degree by introduction of a noncanonical disulfide linkage formed between Cys39 and Cys87, a position they identified by inspecting sdAb crystal structures.12 More recently, Zabetakis et al. explored the effects of introducing four noncanonical disulfide linkages (Cys4‐Cys117, Cys36‐Cys110, Cys54‐Cys78, and Cys53‐Cys111.2) into a highly thermostable llama sdAb.24 They found that all four engineered sdAbs (each bearing a different noncanonical disulfide linkage in addition to Cys23‐Cys104) had modestly elevated T ms and no loss of antigen‐binding affinity, despite three of the linkages involving positions in CDR3. Of the previously described putative noncanonical disulfide linkages spanning variable domain framework residues, only the Cys54‐Cys78 engineered Cys pair has been verified to form a bona fide disulfide linkage by mass spectrometry.14, 21, 22
Two recent studies suggested that the side chains of residues at IMGT positions 40 and 55 of antibody variable domains were positioned favorably for interaction with one another,25 and that the VH domains of rabbit IgGs against HIV‐1 bore Cys residues at these positions, forming a disulfide linkage.26 Thus, the purpose of this study was to examine the effects of introducing an engineered Cys40‐Cys55 disulfide linkage (Fig. 1) on sdAb thermostability, solubility, and antigen binding.
Figure 1.
Locations of canonical and noncanonical disulfide linkages in immunoglobulin variable domains. (A) Primary amino acid sequence of the Clostridium difficile toxin A‐specific VHH A26.828 with the locations of the canonical Cys23‐Cys104 disulfide linkage (cyan) and the noncanonical Cys54‐Cys78 (magenta) and Cys40‐Cys55 (green) disulfide linkages indicated. (B) Crystal structure of A26.8 VHH (PDB ID: 4NBZ) with the locations of canonical and noncanonical disulfide linkages colored as in A.
Results and Discussion
Barthelemy et al. suggested that the side chains of residues at IMGT positions 40/55 and 44/50 of antibody variable domains were favorably positioned for interaction,25 and we thus hypothesized that introduction of Cys pairs at these locations might result in formation of stabilizing intra‐domain disulfide linkages. However, an engineered form of the human VH domain, HVHP430,27 bearing Cys44 and Cys50 residues showed no increase in T m compared with the parent sdAbs (data not shown). We therefore assumed that disulfide linkage formation between the Cys44‐Cys50 pair was not possible and focused our efforts on the putative Cys40‐Cys55 linkage (Fig. 1).
We first attempted to introduce the Cys40‐Cys55 disulfide linkage into a large panel of sdAbs (9 VHHs, 12 VHs, and 11 VLs; Table 1) to examine the effects of this linkage on sdAb thermostability. Our panel consisted of VHHs against Clostridium difficile toxin A28 and human IGF1R,29, 30 the blood–brain barrier‐transmigrating VHH FC5,31 and several previously described autonomous human VH and VL domains.10, 21, 22, 27 Eleven VLs were originally slated for testing, but after introduction of Cys40 and Cys55 residues into their coding sequences, only two showed detectable expression in 1‐L Escherichia coli TG1 cultures. However, all of the disulfide‐engineered VHs and VLs that did express were bound by the generic ligands, protein A and protein L, respectively, indicating correct folding of the immunoglobulin domain (data not shown). We screened the 23 disulfide‐engineered sdAbs (along with their wild‐type counterparts) for potentially increased T ms using a high‐throughput thermal shift assay, and to our surprise, found that incorporation of the Cys40‐Cys55 disulfide linkage enhanced the T ms of all sdAbs tested (data not shown). Thus, we measured T ms using circular dichroism and found that with one exception (HVHP414), each disulfide‐engineered sdAb showed an increase in T m compared with the parent sdAb [Fig. 2(A), Table 1]. The average sdAb T m increase conferred by the Cys40‐Cys55 linkage was 10.0°C (range: 0–21.8°C) and the linkage appeared to have similar effects in all three sdAb types. We identified tryptic peptides from each of 13 representative sdAbs (6 VHHs, 6 VHs, and 1 VL) corresponding to the molecular masses of peptides linked through Cys40/Cys55, confirming the presence of a bona fide disulfide linkage (Supporting Information Table S1). As previously demonstrated for the Cys54‐Cys78 disulfide linkage,14, 21, 22 the mass spectrometry data indicated that in most sdAbs, disulfide linkages were probably formed heterogeneously between the four available Cys residues (Cys23, Cys40, Cys55, and Cys104).
Table 1.
Biophysical Properties of Wild‐Type and Disulfide‐Engineered sdAbs
| sdAb type | sdAb | Disulfide linkage(s) | Mfor (Da)a | MMALS (Da)b | Monomer (%)c | T m (°C) | Fraction refolded |
|---|---|---|---|---|---|---|---|
| VHH | A4.2 | 23–104 | 15727 | n.d. | n.d. | 87.2 ± 0.1 | 0.97 ± 0.01 |
| 23–104, 40–55 | 15806 | 14080 | 91 | 92.2 ± 0.2d | 0.81 ± 0.03 | ||
| A5.1 | 23–104 | 15802 | 14920 | >95 | 78.3 ± 0.4 | 0.91 ± 0.01 | |
| 23–104, 40–55 | 15853 | 15940 | 78 | 86.5 ± 0.2 | 0.80 ± 0.02 | ||
| A20.1 | 23–104 | 15714 | 15940 | >95 | 74.2 ± 0.3 | 0.92 ± 0.02 | |
| 23–104, 40–55 | 15778 | 15500 | >95 | 77.5 ± 0.1 | 0.67 ± 0.01 | ||
| A24.1 | 23–104 | 15767 | 16010 | >95 | 77.0 ± 0.2 | 0.93 ± 0.01 | |
| 23–104, 40–55 | 15860 | 14910 | 63 | 83.7 ± 0.1 | 0.83 ± 0.01 | ||
| A26.8 | 23–104 | 16017 | 14250 | >95 | 82.3 ± 0.3 | 1.00 ± 0.03 | |
| 23–104, 40–55 | 16053 | 14810 | 62 | 90.1 ± 1.2 | 0.86 ± 0.02 | ||
| IGF1R3 | 23–104 | 16215 | 14870 | >95 | 74.6 ± 0.4 | 0.11 ± 0.08 | |
| 23–104, 40–55 | 16277 | 14980 | 90 | 82.3 ± 1.1 | 0.69 ± 0.01 | ||
| IGF1R4 | 23–104 | 15733 | 13970 | >95 | 81.3 ± 0.1 | 0.97 ± 0.01 | |
| 23–104, 40–55 | 15746 | 13630 | 61 | 85.9 ± 0.1 | 0.88 ± 0.02 | ||
| IGF1R5 | 23–104 | 15747 | 15130 | >95 | 61.9 ± 0.5 | 0.84 ± 0.01 | |
| 23–104, 40–55 | 15781 | 14070 | >95 | 73.4 ± 0.1 | 0.72 ± 0.02 | ||
| FC5 | 23–104 | 15514 | 14050 | >95 | 67.2 ± 0.1 | 0.87 ± 0.02 | |
| 23–104, 40–55 | 15507 | 14960 | 92 | 85.8 ± 0.7 | 0.75 ± 0.01 | ||
| VH | HVHP414 | 23–104 | 15105 | 14600 | 80 | 58.8 ± 0.1 | 0.80 ± 0.01 |
| 23–104, 40–55 | 15068 | 15930 | 79 | 58.6 ± 0.1 | 0.33 ± 0.01 | ||
| HVHP421 | 23–104 | 15392 | 15220 | 69 | 58.8 ± 0.3 | 0.35 ± 0.02 | |
| 23–104, 40–55 | 15440 | 15660 | 88 | 74.4 ± 0.5 | 0.22 ± 0.06 | ||
| HVHP429 | 23–104 | 15097 | 15110 | 88 | 60.6 ± 0.1 | 0.78 ± 0.00 | |
| 23–104, 40–55 | 15115 | 14910 | 24 | 67.4 ± 0.1 | 0.53 ± 0.01 | ||
| HVHP44 | 23–104 | 15411 | 14720 | 93 | 66.1 ± 0.1 | 0.54 ± 0.13 | |
| 23–104, 40–55 | 15459 | 16380 | 97 | 79.0 ± 0.5 | 0.34 ± 0.06 | ||
| HVHP419 | 23–104 | 15084 | n.d. | n.d. | 59.2 ± 0.5 | 0.11 ± 0.01 | |
| 23–104, 40–55 | 15133 | 15390 | 94 | 73.2 ± 0.1 | 0.04 ± 0.04 | ||
| HVHB82 | 23–104 | 14278 | 17080 | 53 | 61.2 ± 0.2 | 0.39 ± 0.09 | |
| 23–104, 40–55 | 14326 | 14390 | >95 | 78.7 ± 0.1 | 0.11 ± 0.06 | ||
| HVHP423 | 23–104 | 15103 | n.d. | n.d. | 57.0 ± 2.0 | 0.17 ± 0.17 | |
| 23–104, 40–55 | 15123 | 16710 | >95 | 67.8 ± 0.0 | 0.21 ± 0.05 | ||
| HVHM41 | 23–104 | 15470 | 16360 | 47 | 52.8 ± 2.2 | 0.39 ± 0.21 | |
| 23–104, 40–55 | 15518 | 15630 | 28 | 74.6 ± 0.1 | 0.51 ± 0.06 | ||
| HVHM81 | 23–104 | 15195 | 15550 | 95 | 69.6 ± 0.4 | 0.86 ± 0.07 | |
| 23–104, 40–55 | 15207 | 15020 | 94 | 80.3 ± 0.1 | 0.79 ± 0.04 | ||
| HVHP428 | 23–104 | 15675 | 15470 | 94 | 65.7 ± 0.2 | 0.00 ± 0.01 | |
| 23–104, 40–55 | 15647 | 15310 | 92 | 80.1 ± 0.4 | 0.46 ± 0.03 | ||
| HVHP420 | 23–104 | 15069 | 14390 | 79 | 58.0 ± 0.1 | 0.75 ± 0.05 | |
| 23–104, 40–55 | 15018 | 14710 | 80 | 67.1 ± 0.2 | 0.83 ± 0.02 | ||
| HVHP413 | 23–104 | 15177 | n.d. | n.d. | n.d. | n.d. | |
| 23–104, 40–55 | 15149 | 15520 | 43 | 70.7 ± 1.0 | 0.50 ± 0.05 | ||
| VL | HVLP335 | 23–104 | 13904 | 13970 | >95 | 63.6 ± 0.1 | 0.95 ± 0.04 |
| 23–104, 40–55 | 13876 | 12950 | >95 | 71.7 ± 0.9 | 0.74 ± 0.05 | ||
| HVLP342 | 23–104 | 13950 | 13770 | >95 | 61.6 ± 0.1 | 1.00 ± 0.14 | |
| 23–104, 40–55 | 13878 | 13340 | >95 | 67.2 ± 0.3 | 0.73 ± 0.06 |
n.d., not determined.
Calculated from the sdAb primary amino acid sequence including c‐Myc and His6 tags.
Calculated for the monomeric sdAb peak area from SEC‐MALS data.
Calculated from SEC‐MALS data.
Minimum estimate.
Figure 2.
Effects of the noncanonical Cys40‐Cys55 disulfide linkage on sdAb thermostability and other properties. (A, B) Changes in sdAb T m and fraction refolded (α) following introduction of the Cys40‐Cys55 linkage were measured by circular dichroism. (C) Changes in sdAb monomericity following introduction of the Cys40‐Cys55 linkage were measured by SEC‐MALS. The lower monomericity of some wild‐type VH domains compared with previous studies was likely due to long‐term storage at 4°C. (D) Perturbations in the K Ds of antigen‐specific VHHs following introduction of the Cys40‐Cys55 linkage were assessed by SPR. Medians/averages are indicated by a red dash.
However, we also found that the characteristic reversible thermal unfolding of sdAbs was negatively affected by introduction of the Cys40‐Cys55 disulfide linkage. After an initial melt and return to room temperature, most disulfide‐engineered sdAbs (8/9 VHHs, 8/11 VHs, and 2/2 VLs) had smaller refolded fractions than the parent sdAbs available for the second melt [Fig. 2(B), Table 1]. This finding may be related to incomplete and heterogeneous formation of disulfide linkages in the engineered sdAbs, with free thiols becoming oxidized in the denatured state and preventing refolding. In most cases, impairment of reversible thermal unfolding was relatively minor (average Δα = −0.08; range: −0.47 to 0.58). We also used SEC‐MALS to examine the effects of the Cys40‐Cys55 disulfide linkage on sdAb monomericity and found that some sdAbs bearing this linkage had higher propensities to form soluble aggregates than the parent sdAb [Fig. 2(C), Table 1]. Approximately half (9/19) of sdAbs tested showed significantly (>10%) decreased monomericity upon introduction of the Cys40‐Cys55 disulfide linkage, with the remainder tolerating it with no adverse effects. Finally, we assessed the impact of the Cys40‐Cys55 disulfide linkage on binding of the eight antigen‐specific sdAbs (all VHHs) among our panel. We found that for 6/8 VHHs, introduction of the Cys40‐Cys55 disulfide linkage resulted in significantly (≥10‐fold increase in K D) weaker affinity for cognate antigen [Fig. 2(D), Table 2].
Table 2.
Effects of the Cys40‐Cys55 Disulfide Linkage on Binding of Antigen‐Specific VHHs by SPR
| VHH | Disulfide linkage(s) | k on (M−1 s−1) | k off (s−1) | K D (nM) |
|---|---|---|---|---|
| A4.2 | 23–104 | 1.5 × 106 | 4.9 × 10−2 | 33 |
| 23–104, 40–55 | 8.8 × 105 | 5.1 × 10−2 | 58 | |
| A5.1 | 23–104 | 2.5 × 106 | 7.2 × 10−3 | 3 |
| 23–104, 40–55 | 2.3 × 104 | 4.8 × 10−3 | 208 | |
| A20.1 | 23–104 | 2.5 × 106 | 3.2 × 10−3 | 1 |
| 23–104, 40–55 | 6.5 × 105 | 7.2 × 10−3 | 11 | |
| A24.1 | 23–104 | 3.0 × 104 | 2.4 × 10−3 | 80 |
| 23–104, 40–55 | 1.1 × 105 | 3.0 × 10−2 | 273 | |
| A26.8 | 23–104 | 1.5 × 106 | 2.8 × 10−2 | 19 |
| 23–104, 40–55 | ‐ | ‐ | 178a | |
| IGF1R3 | 23–104 | 3.5 × 105 | 3.4 × 10−4 | 1 |
| 23–104, 40–55 | 7.8 × 103 | 1.5 × 10−3 | 192 | |
| IGF1R4 | 23–104 | 1.1 × 106 | 2.6 × 10−4 | 0.2 |
| 23–104, 40–55 | ‐ | ‐ | 75a | |
| IGF1R5 | 23–104 | 4.2 × 105 | 5.2 × 10−4 | 1 |
| 23–104, 40–55 | ‐ | ‐ | 36a |
Biphasic binding was observed at higher concentrations resulting in a poor fit to a 1:1 binding model, and thus estimates of steady‐state affinities are reported.
In summary, we showed that introduction of a noncanonical disulfide linkage spanning IMGT positions 40 and 55 can confer increased thermostability to sdAbs, at the expense of (i) minor loss of reversible thermal unfolding, (ii) increased aggregation in approximately half of sdAbs tested, and (iii) major loss of antigen‐binding affinity in approximately three quarters of sdAbs tested. We presume that stabilization by the Cys40‐Cys55 disulfide linkage, as well as potential loss of solubility and antigen binding, would extend to the VH and VL domains of Fabs and full‐length IgGs. We did not assess whether the Cys40‐Cys55 disulfide linkage imparts chemical or protease resistance to sdAbs, as has been shown for other noncanonical disulfide linkages. A summary of the impact of the Cys40‐Cys55 linkage and other known noncanonical disulfide linkages on sdAb properties is shown in Table 3 (only linkages involving non‐CDR positions are shown). Disregarding the fact that the monomericities and antigen‐binding affinities of disulfide‐engineered sdAbs were not measured accurately (or at all) in every previous study, the available data currently suggest that compared with the Cys40‐Cys55 disulfide linkage, sdAb stabilization through introduction of a Cys54‐Cys78 disulfide linkage results in a similar increase in T m and is less likely to impair sdAb solubility or antigen recognition. This greater impairment of antigen binding of the Cys40‐Cys55 linkage compared with the Cys54‐Cys78 linkage is perhaps to be expected, given the positions of Cys 40 and Cys 55 immediately adjacent to the CDR1 C‐terminus and the CDR2 N‐terminus, respectively (Fig. 1). However, individual sdAbs may differ in their tolerance of specific noncanonical disulfide linkages and this must be tested empirically. Alternatively, noncanonical disulfide linkages can be introduced into synthetic sdAb libraries a priori to avoid the negative consequences of post hoc disulfide engineering.32
Table 3.
Impact of Noncanonical Disulfide Linkage Position on sdAb Thermostability and other Properties
| Disulfide linkage | n | ΔT m (°C) | Impaired solubilitya | Impaired bindingb | Reference |
|---|---|---|---|---|---|
| Cys54–Cys78 | 1 | 10 | n.d. | 0/1 sdAb (0%) | 11 |
| 3 | 11.4 (6.2–18.8) | 0/3 sdAbs (0%) | 0/3 sdAbs (0%) | 12 | |
| 6 | 7.3 (2.1–11.6) | 0/6 sdAbs (0%) | 2/6 sdAbs (33%) | 14 | |
| 1 | 17c | n.d. | 0/1 sdAb (0%) | 13 | |
| 4 | 5.5 (3.4–12.0) | n.d. | n.d. | 17 | |
| 1 | 17 | n.d. | 0/1 sdAb | 16 | |
| 2 | 10 (9–11) | n.d. | 0/2 sdAbs | 18 | |
| 3 | 16 (15–17)d | n.d. | 1/3 sdAbs | 19 | |
| 8 | 11.1 (6–19) | n.d. | n.d. | 20 | |
| 2 | 12.7 (7.9–17.4) | 0/2 sdAbs | 0/2 sdAbs | 15 | |
| 4 | 16.0 (13.9–17.6) | 0/4 sdAbs (0%) | n.d. | 21 | |
| 8 | 11.8 (5.4–17.3) | 1/8 sdAbs (13%) | n.d. | 22 | |
| 1 | ≥6 | n.d. | 0/1 sdAb (0%) | 24 | |
| Pooled average: | 11.2 (2.1–19) | 1/23 sdAbs (4%) | 3/20 sdAbs (15%) | ||
| Cys39–Cys87 | 3 | 9.5 (4.2–17.8) | 0/3 sdAbs (0%) | 1/3 sdAbs (33%) | 12 |
| Cys40–Cys55 | 23 | 10.0 (0–21.8)e | 9/19 sdAbs (47%) | 6/8 sdAbs (75%) | This study |
n.d., not determined.
Defined as ≥10% soluble aggregate.
Defined as ≥10‐fold increase in K D.
Measured at pH 5.5.
The disulfide‐engineered sdAbs bore minor FR sequence changes in addition to the two Cys residues.
Only 2/11 VLs tested tolerated this disulfide linkage and could be expressed.
Materials and Methods
Materials and reagents
Ampicillin, isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) and SYPRO Orange were from Sigma‐Aldrich (St. Louis, MO). HisTrap HP affinity columns were from GE Healthcare (Piscataway, NJ). Mini‐PROTEAN TGX 4–20% stain‐free sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels were from Bio‐Rad (Hercules, CA). Recombinant human IGF1R ectodomain (Cat. No. 391‐GR‐050) was from R&D Systems (Minneapolis, MN). C. difficile toxin A was from List Biological Laboratories, Inc. (Campbell, CA). Recombinant protein A and protein L were from Thermo‐Fisher Scientific (Waltham, MA).
Expression and purification of sdAbs
The coding sequences of sdAbs were synthesized and cloned into the pSJF2H expression vector by GenScript USA (Piscataway, NJ). C‐terminally c‐Myc‐ and His6‐tagged sdAbs were expressed in the periplasm of E. coli TG1 cells and purified by immobilized metal ion affinity chromatography (IMAC) as previously described.10, 32, 33 Briefly, competent E. coli TG1 cells were transformed with each expression vector, and 10 mL of 2×YT media containing 100 μg/mL ampicillin were inoculated with a single colony and grown overnight at 37°C with 220 rpm shaking. The following day, 5 mL of the overnight culture was subcultured into 250 mL of 2×YT media containing 100 μg/mL ampicillin and grown at 37°C with 250 rpm shaking. When the culture reached an OD600 of 0.5, expression was induced with 1 mM IPTG. After overnight growth, periplasmic proteins were extracted by osmotic sucrose shock and sdAbs were purified by IMAC on an ÄKTA FPLC protein purification system (GE Healthcare). The purity and integrity of sdAbs were assessed by reducing and nonreducing SDS‐PAGE.
Circular dichroism
T ms were determined by circular dichroism as described previously10, 14, 21 using a Jasco J‐815 spectropolarimeter equipped with a Peltier thermoelectric‐type temperature control system (Jasco, Easton, MD). All sdAbs were adjusted to 100 μg/mL in 100 mM sodium phosphate buffer, pH 7.4, and ellipticity was measured as a function of temperature at wavelengths between 205 and 210 nm. After the first thermal denaturation, sdAbs were cooled to 25°C for 60 min at room temperature and denaturation was repeated to calculate refolding efficiencies, expressed as α‐values. All circular dichroism measurements were made in duplicate and reported as means and standard deviations.
Thermal shift assay
Thermofluor assays were conducted essentially as previously described.10 SYPRO Orange was diluted 1:100 from the 5,000× stock in phosphate‐buffered saline, pH 7.4, containing 1 mM EDTA. Two microliters of diluted SYPRO Orange was mixed with 18 μL of sdAb (0.25 mg/mL) in white low‐profile PCR tubes with optical flat caps. Using an iQ 5 real‐time PCR system (Bio‐Rad), the temperature was ramped at 1°C/min from 30°C to 95°C and fluorescence was measured at 0.5°C intervals. The wavelengths for excitation and emission were 490 and 575 nm, respectively. T ms were calculated as the temperature at which the maximum rate of change in fluorescent signal (d(RFU)/dt) was observed.
Size exclusion chromatography (SEC) and SEC‐MALS
Analytical and preparative SEC were conducted by injecting sdAbs over a Superdex 75 GL column (GE Healthcare) connected to an ÄKTA FPLC protein purification system. The mobile phase consisted of either HBS‐EP [10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA, and 0.005% (v/v) surfactant P20] or HBS‐EP+ (10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). UPLC‐SEC‐MALS analyses were conducted essentially as previously described10, 22 using an Acquity BEH‐125 column (Waters, Milford, MA) connected to an Acquity UPLC H‐Class Bio system (Waters) with miniDAWN MALS detector and Optilab UT‐rEX™ refractometer (Wyatt Technology, Santa Barbara, CA). Data were processed using ASTRA 6.1 software (Wyatt).
Mass spectrometry
Approximately 50 μg of each sdAb were digested with trypsin as previously described14 and digestion was verified by SDS‐PAGE. The trypsin‐cleaved sdAbs were desalted by electrodialysis, equilibrated in 50% acetonitrile/0.1% formic acid and analyzed by nanoRPLC‐ESI‐MS with data‐dependent analysis using a nanoAcquity UPLC system coupled to a Q‐TOF II Ultima hybrid quadrupole/TOF mass spectrometer (Waters). Proteolytic peptides were first loaded onto a 180 μm I.D. × 20 mm 5‐μm Symmetry C18 trap (Waters) in solvent A (0.1% formic acid), then eluted onto a 100 μm I.D. × 10 cm 1.7‐μm BEH130C18 column (Waters) using linear gradients from 0% to 36% solvent B (acetonitrile/0.1% formic acid) over 36 min and 36–90% solvent B over 2 min. Peptide MS2 spectra were searched against the relevant sdAb sequence using the Mascot search engine (Matrix Science, London, UK). MS2 spectra of disulfide‐linked peptides were deconvoluted using the MaxEnt 3 program (Waters) for de novo sequencing.
Surface plasmon resonance (SPR)
For antigen‐specific sdAbs, the monovalent affinities of wild‐type and disulfide‐engineered sdAbs for their cognate antigens were determined by SPR. All SPR analyses were conducted at 25°C using either a Biacore 3000 instrument or a Biacore T200 instrument (GE Healthcare). Prior to SPR experiments, monomeric sdAbs were purified by preparative SEC into HBS‐EP for Biacore 3000 experiments (IGF1R binding) and HBS‐EP+ for Biacore T200 experiments (C. difficile toxin A binding, protein A binding, and protein L binding). Approximately 2300, 10,000, 1200, and 600 resonance units, respectively, of human IGF1R ectodomain, C. difficile toxin A, protein A, and protein L were immobilized on CM5 or CM5 Series S sensor chips in 10 mM acetate buffer, pH 4.0–4.5, using an amine coupling kit (GE Healthcare). An ethanolamine‐ or ovalbumin‐blocked flow cell was used as reference. Monomeric sdAbs (at least six concentrations, ranging from 0.25 nM to 3 μM depending on the sdAb) were injected over the relevant antigen surface at a flow rate of 20 μL/min with contact times of 120–300 s and dissociation times of 300–600 s. Binding to IGF1R and toxin A was analyzed using multi‐cycle kinetic analysis and binding to protein A and protein L was analyzed using steady‐state affinity analysis. The surfaces were regenerated using a 6‐s pulse of 10 mM glycine, pH 2.0. All data were analyzed by fitting to a 1:1 interaction model using BIAevaluation 4.1 software (GE Healthcare).
Conflict of Interest
The authors have no competing interests to declare.
Supporting information
Appendix S1: Supporting Information
Figure S1
Acknowledgments
The authors gratefully acknowledge Camille Hébert‐Martineau and Henk van Faassen for excellent technical assistance. The authors thank Joe Schrag for help with SEC‐MALS experiments.
Dae Young Kim's current address is New Drug Development Center, Osong Medical Innovation Foundation, South Korea.
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Supplementary Materials
Appendix S1: Supporting Information
Figure S1