KEYWORDS: Antibody complex, antibody selection, conformational change, mechanism of action, Tm-shift; toxins, unfolding

Structure of the A3C8 single-domain antibody

The A3C8 sdAb was first crystallized. Data collection and refinement statistics are shown inTable 1. A3C8 was found to be monomeric using a calibrated gel-filtration column (Table 2). The structure of the A3C8 sdAb (PDB 5SV4) was used to solve the structure of the sdAb-RTA1-33/44-198 complex by molecular replacement.

Structure of the A3C8:RTA1-33/44-198 complex

Previously, we crystallized 2 thermostabilized variants of RTA1-33/44-198 containing disulfide bonds (Fig. 1);³⁰ however, we were not able to crystallize the parent protein, RTA1-33/44-198, alone. RTA1-33/44-198 and its variants contain a deletion of loop residues 34–43; these hydrophobic loop residues pack with the C-terminal 199–267 residues (Fig. S1). The removal of residues 34–43 and 199–267 was previously shown to increase the T_m by 8 degrees compared with RTA.^21,37

Co-crystallization of RTA1-33/44-198 with sdAb A3C∼8 enabled us to solve the first structure of the original RTA1-33/44-198 variant and examine the conformation of the sdAb-bound antigen. When sdAb-bound RTA1-33/44-198 was compared with the disulfide bonded variants (PDB 3MK9 and 3LC9),³⁰ conformational differences were observed in 3 regions: residues 33–52; residues 131–136 and residues 182–188 (Fig. S2). The differences in residues 33–52 are likely due to the disulfide bonds that were incorporated at R48C/T77C and V49C/E99C. In the sdAb A3C8:RTA1-33/44-198 complex, the conformation of the loop residues 131–136 was more similar to that of RTA in the ricin holotoxin (PDB 2AAI), than to the conformation found in the disulfide-bonded variants (PDB 3LC9, 4IMV, and 3MK9).^30,21 In the structures of the disulfide variants, the conformation of loop residues 131–136 found in RTA (PDB 1J1M)³⁸ would clash with an adjacent molecule at the crystal packing interface. We had previously shown that the B-factors of residues 131–136 were higher in the disulfide-bonded variants compared with RTA, and that the density for the loop was poor, suggesting static or dynamic disorder of these residues.³⁰ In the A3C8:RTA1-33/44-198 complex (PDB 5SV3), these residues also were involved in a crystal packing interface, but had well-defined density while residues 188–198 (near the 33–44 loop) appeared disordered. The C-terminal domain packs with the 131–136 loop residues and residues 182–188. The C-terminal domain and residues 182–188 contact RTB (Fig. 1, Fig. S1, S2). The conformation of these loop residues may be stabilized by the C-terminal domain residues 199–267 or by sdAb A3C8 binding. However, a structure of the free RTA1-33/44-198 is still needed to confirm this effect.

Effects of the Q98A, Q112A and R114A mutations on sdAb A3C8 binding

The epitope recognized by A3C8 was initially identified using the crystal structure of the complex. The epitope then was confirmed by site-directed mutagenesis, Western blotting, and surface plasmon resonance (SPR;Fig. 2).

A C-terminal His-tagged sdAb and anti-His-tag IgG were used to detect RTA and its variants in Western blot analysis. Antigens were separated by SDS-PAGE. Based upon the crystal structure (PDB 5SV3), 3 residues were selected for mutation: Q98A, Q112A, and R114A. Other previously characterized disulfide-bonded RTA1-33/44-198 variants^30,21 were included: R48C/T77C; V49C/E99C; A90C/V111C; R125C/A165C; E138C/P143C; N141C/I192C; and R48C/T77C/D75N. A total of 10 RTA1-33/44-198 variants were examined in addition to recombinant RTA and RTA purified fromRicinus communis. Staining was observed for all of the RTA1-33/44-198 variants (0.5 µg per lane) with the exception of R114A, indicating that R114A mutation disrupted sdAb A3C8 binding. The Q112A variant showed slightly reduced staining.

SPR was used to examine changes in affinity to the epitope since multiple contacts were observed between the antibody and antigen (Table 3). For sdAb A3C8, the R114A mutation led to the most significant increase in the dissociation constant (49-fold higher K_d), and the Q112A mutation lead to a ˜2-fold higher K_d compared with RTA1-33/44-198.

Ab binding to the denatured RTA was consistent with binding to a linear epitope (Fig. 2A). However, binding to an N-terminally biotinylated-peptide that contains residues H94-T116 (⁹⁴HPDNQEDAEAITHLFTDVQNRYT¹¹⁶) and the UNIVAX R70 epitope (underlined) was not detectable by SPR, suggesting that the Ab may make an important contact to residues outside of this region. A3C8 carries the same complementarity-determining region (CDR) sequences as C8, but has a different framework. A3C8 bound less well to RTA than C8 (Table 3) suggesting that the framework residues may favor particular side-chain conformations of CDR residues involved in binding or alter the mobility of the CDRs.

Epitope mapping of other anti-ricin sdAb: D12f, D10, E1, F6, C10neg and H1W12V

We also identified the general locations of the epitopes of the other sdAb tested. The K_d values measured for RTA, RiVax, RTA1-33/44-198, and the 3 variants are summarized inTable 3. The structure of the D10:RTA complex had been previously determined,²² and the Q98A, Q112A and R114A mutations did not have significant effects on binding affinity consistent with the structure (Fig. 3E). Like sdAb A3C8, the D12f sdAb had markedly reduced affinity (41-fold higher K_d) for the R114A variant, suggesting that the epitopes of the neutralizing D12f and A3C8 sdAb overlapped (n.b. sdAb A3C8 and C8 contain the same CDRs).

The H1W12V Ab, which is non-neutralizing, had significantly reduced affinity for RTA1-33/44-198 (>100-fold higher K_d) and the 3 RTA1-33/44-198 variants, suggesting that the epitope is within the C-terminal domain residues, 199–267, or within the loop (residues 34–43). The loop (residues 33–44) packs with the C-terminal domain³⁷ (Fig. S1). The weakly neutralizing G11 sdAb, for which a structure is known, is an example of an sdAb that can bind the C-terminal domain²² (Fig. 3E). The non-neutralizing Ab, C10neg, did not bind to RTA alone. This Ab is thought to bind to regions of both the A- and B-chains of ricin. The C-terminal domain of RTA packs with RTB (Fig. 1). The F6 sdAb, which also is a non-neutralizing Ab, had slightly reduced binding affinity to RTA1-33/44-198 (5-fold), and showed no significant binding to the R114A variant, suggesting that the F6 epitope may be near that of sdAb A3C8. The epitope of F6 may be near the epitope recognized by the non-neutralizing sdAbs A7 and G12.²² The E1 sdAb, which is also non-neutralizing, was not significantly affected by the Q98A, Q112A or R114A mutations or by the C-terminal domain truncation.

Analysis of linear regression models and comparison of mean values

We assembled a set of neutralizing and non-neutralizing Abs. The sdAb was classified as neutralizing if >50% cell viability was obtained in the cell-based assays using the specified concentrations of ricin and sdAb. In cell-based assays using the ricin holotoxin, 50% cell viability corresponded to sdAb with IC₅₀ values ≤ 80 nM. The sdAb were classified as non-neutralizing if <50% cell viability was obtained or the Ab had IC₅₀ values > 80 nM. For the 4 non-neutralizing sdAb used in this study, the IC₅₀ values exceeded the highest concentration of ricin tested (5 µM) (Table 4). The A3C8 sdAb is an engineered thermostabilized variant containing the CDRs of the C8 sdAb and the framework regions of a thermostable anti-SEB (Staphylococcal enterotoxin B) Ab called A3 (T_m = 85°C).³⁹ The A3C8 sdAb has a T_m that is 10°C higher than that of C8. The A3C8 variant binds the same epitope as C8, but with 3-fold lower affinity than C8 (Table 2). This difference allowed us to examine the effects of K_d. The binding of D12f was affected by the R114A mutation and its epitope likely overlaps with that of C8 and A3C8. SdAb D10 is also a neutralizing Ab; its epitope is known and differs from that of A3C8, C8, and D12f (Fig. 3). For non-neutralizing Abs, we selected H1W12V, C10neg, F6 and E1. The E1 and H1W12V sdAbs recognize an epitope within the C-terminal domain residues 199–267, and the C10neg sdAb recognizes both RTA and RTB. To find correlations between the toxin-neutralizing activities of the sdAb and various biophysical parameters, we performed linear regression analysis and examined the r²-values (Fig. 5). We also compared the difference between the mean values of the biophysical parameters relative to the variation in the sample data, and p-values were calculated from T-values. In our cell viability assays, all of the sdAb concentrations were above the K_d values (i.e., stoichiometric binding regime). All experiments were carried out in triplicate, and the averages and standard deviations are summarized inTable 4. Melting temperatures were measured by monitoring changes in secondary structure as the temperature was raised using a CD spectrometer (Table 2,Fig. 4).

Cell viability vs. percent inhibition of translation

We observed a correlation between cell viability vs. percent inhibition of translation (r² = 0.912). The T-value was 6.5747 and p-value 0.000297; however, the correlation is not necessarily related to the MOA of anti-RTA Abs (Fig. 5A). This coincidence has been noted by others,⁴ and may be due to the location of active site and substrate binding residues within the neutralizing epitopes⁴⁰ (Fig. S3). Residues 90–110 contain the UNIVAX R70 epitope (N97-F108) and form a portion of the substrate binding site. Similarly, the B-cell epitope (L161-I175) forms a portion of the substrate binding site. However, while ricin can enter into the Golgi, ER and cytoplasm,^41,42 the anti-ricin Abs likely do not unfold and translocate into the cytoplasm via retrograde transport (i.e., ERAD system) to inhibit the enzymatic activity of RTA. Antibodies are typically separated from their cargo and recycled via the neonatal Fc receptor. Notably, a non-neutralizing anti-RTA antibody known as mAb RAC23 that can inhibit RTA's enzymatic activity, but fails to protect in cell-based assays, has been reported.^4,43,44 RAC23 actually enhanced the toxicity of ricin in a mouse model.⁴ The neutralizing anti-RTA mAb called RAC18 led to the accumulation of ricin at the cell surface, suggesting that this neutralizing anti-RTA Ab halts an early step(s) in retrograde transport,⁴¹ rather than RTA substrate binding or catalysis in the cytoplasm. Comparison of sdAb A3C8 with C8 showed that both sdAb comparably inhibited cell-free translation, and that inhibition in cell-free translation could not distinguish between the 2 sdAb. So, while neutralizing and non-neutralizing Abs could be distinguished from one another in this graph, a MOA involving the inhibition of the enzymatic activity in the cytoplasm by an Ab is not supported by Abs such as RAC23.⁴¹

T_m shift vs. cell viability

A strong correlation between cell viability and the magnitude of the T_m shift was observed for neutralizing Abs and yielded a coefficient of determination r² = 0.992 and a linear relationship with a positive slope of 0.65 ± 0.05 (ΔT_m/Δ% cell viability) and a negative y-intercept (−26 ± 3); the x-intercept corresponded to 40% cell viability and was near the 50% cutoff for neutralizing vs. non-neutralizing Abs (Fig. 5B). A similar correlation was obtained with IC₅₀ values measured by cell-based assays and T_m shifts (r² = 0.846) (Fig. 5D). The ranges on the x- and y-axes were relatively large compared with the other parameters, and the points were well dispersed along the line. Excluding the engineered A3C8 sdAb, the mean values of the T_m-shifts for the neutralizing vs. non-neutralizing Abs were compared and produced a T-value = 6.48 and a p-value of 0.00146 (one-tailed), showing that the magnitude of the T_m-shifts for the neutralizing Abs was significantly higher than those of the non-neutralizing Abs. With the inclusion of A3C8, the T-value was 3.15 and the p-value was 0.0127 (one-tailed), showing that the mean value of the T_m-shifts obtained for the neutralizing Abs still was significantly different from that of the non-neutralizing Abs. Thus, the magnitude of the T_m-shift could be used to distinguish between neutralizing and non-neutralizing Abs. Notably, the magnitude of the T_m-shift rank-ordered the sdAb in the same manner as the cell-based assay data.

The apparent T_m values of the Ab-Ag complexes containing RTA (residues 1–267, 30 kDa) were well correlated with the ability of the sdAb to protect cells from intoxication during exposure to the 59 kDa A-B ricin toxin. However, a much poorer correlation was observed as the stability of the ricin A-chain antigen was altered (i.e., RTA1-33/44-198 was used in place of RTA), suggesting that a property of the Ab-Ag complexes had been perturbed (Fig. 4). No significant correlation between cell viability and T_m-shift was observed with the thermostabilized RTA1-33/44-198 variant (r² = 0.2836). RTA1-33/44-198 had been engineered to have a reduced propensity to unfold, and lacks the C-terminal domain and a loop formed by residues 34–43 that pack with the C-terminal domain. The C-terminal domain packs with RTB³⁷ (Fig. 1, Fig. S2).

T_m shift vs. percent inhibition of translation

Analysis of all of the data points for both neutralizing and non-neutralizing sdAb. This resulted in only a weak correlation (r² = 0.667) between these parameters (Table 4).

T_m shift vs. K_d

For neutralizing and non-neutralizing Abs, no correlation between K_d and T_m shift was observed (r² = 0.295) (Table 4). The T_m reflects a protein's propensity to unfold. This result suggested that the location(s) at which the sdAb bind, rather than their binding affinity, may affect RTA's propensity to unfold. Previously, we had identified 3 regions that became disordered during the early stages of unfolding as the protein was heatedin silico³⁰ (Fig. 3B). We showed that we could elevate the T_m of RTA1-33/44-198 by disulfide bonding these regions to the core of the protein or to one another. The T_m values of 2 disulfide-bonded RTA1-33/44-198 variants were increased by 5 degrees (Fig. 3C, 3D). Disulfide bonds introduced at other locations did not significantly increase the T_m of the RTA1-33/44-198 construct.³⁰ Other groups have empirically identified regions that affect unfolding by mutagenesis alone.²⁷ In regards to Ab binding, residues within these 3 loop regions were found to be engaged by neutralizing Abs (Fig. 3E). Non-neutralizing Abs recognizing so-called “decoy” epitopes adjacent to these regions did not pack well with the 90–110 or 140–170 residues compared with the neutralizing A3C8 (PDB 5SV3) and E5 (PDB 4LGP)²² sdAb. The exposed surface area of these 3 regions differed in the neutralizing vs. non-neutralizing Ab complexes (Fig. 3F).

Cell viability vs. K_d

For RTA and anti-RTA Abs, the K_d was not a strongly predictive parameter of neutralizing activity (Fig. 5C). However, neutralizing activity was affected by K_d in some cases (e.g., C8 vs. A3C8). The K_d value has been shown to be a predictive parameter for other antibodies,³ and the correlation between K_d and protection may largely depend upon the MOA of the protective Ab. From competition experiments we know that D12f and C8 bind the same epitope or overlapping epitopes (Table 3). The K_d values for D12f and C8 differ by a factor of 10; however, the percentage of viable cells obtained for the weaker binding C8 sdAb was higher than that of the tighter binding D12f sdAb. The trend was reversed for A3C8 and C8. A3C8 and C8 have the same CDR sequences and Kd values that differ by 3-fold. A lower percentage of viable cells was obtained with the weaker binding A3C8 sdAb compared with the tighter binding C8 sdAb. The p-value confirmed that K_d alone could not be used to distinguish between neutralizing and non-neutralizing anti-RTA sdAb. For the neutralizing and non-neutralizing sdAb, the T-value was −1.6554 and the p-value was 0.07937 (one tailed). The spread and overlap are shown in (Fig. 5C).

T_m of the antibody vs. T_m of the complex

Thermostabilized Abs have been developed as robust affinity reagents for refrigeration-free use.^45,46,39 Thus, we examined whether a high T_m Ab would be advantageous in preventing the unfolding of its cognate antigen as temperature was varied. Interestingly, there was no advantage in using a thermostabilized sdAb. This was most clearly demonstrated with sdAb A3C8 and C8. SdAb A3C8 has a T_m that is ∼10 degrees higher than that of sdAb C8. The T_m of the A3C8:RTA complex was ∼11 degrees lower than the T_m of the C8:RTA complex. The T_m of the Ab did not correlate with the T_m of the RTA-sdAb complex for neutralizing (r² = 0.064) or non-neutralizing Abs (r² = 0.361). The Tm values of the free neutralizing and non-neutralizing Abs were similar (p-value = 0.30), while the Tm values of the Ab-Ag complexes differed (Tables 2, 4).

Effect of pH on the sdAb-RTA complexes

After the A-B ricin toxin is endocytosed, RTA is separated from RTB shortly (∼minutes) after internalization.⁴⁷ Endosomes play an important role in transporting and sorting molecules to different compartments in the cell.⁴⁸ Endosomes acidify as they transit through the cell and Abs release their antigens. Crystal structures of RTA have been determined at both low (pH 4.2)⁴⁹ and high (pH 8.9)⁵⁰ pH indicating that RTA can remain stably folded in both acidic and basic conditions. To determine if the neutralizing sdAb still could remain bound and reduce RTA's propensity to unfold at low pH, we measured the Tm values of the purified RTA-sdAb complexes at pH 4.5 to mimic the endosomal environment. At pH 4.5 micromolar concentrations of the RTA:A3C8, RTA:C8, RTA1-33/44-198:A3C8 and RTA1-33/44-198:C8 complexes still could be purified by gel-filtration. The K_d values were ∼10–110-fold higher at pH 4.5 than at pH 7.4 (Table 2). The shifts in the T_m of the RTA:A3C8 vs. RTA:C8 complexes still were detectable at pH 4.5 (6.1 degrees and 6.7 degrees, respectively) (Table 2). The elution volumes differed for the sdAb, RTA, and the RTA-sdAb complexes, and were additional confirmation that the complexes had been purified (Fig. S4). For sdAb C8, the magnitude of the T_m shift decreased significantly from 20.3 °C (pH 7.4) to 6.7 °C (pH 4.5), suggesting that the sdAb may predominantly exert their effects at ˜neutral pH, but can remain bound to the antigen at low pH. The stability of the sdAb-RTA complexes at both pH may be important to their MOA.

Cloning of constructs

The sdAb sequences utilized in this work were derived from the variable domain of heavy chain Abs naturally produced by immunized llamas (C8, D12f, H1W12V, C10neg)^33,35 or immunized alpacas (D10, F6, E1).⁵⁷ The cloning of sdAb A3, an anti-SEB Ab with a high T_m (T_m = 85°C),^58,39 was previously described and similar methods were used to construct the other sdAb clones. To construct the engineered A3C8 sdAb variant, the CDRs of the anti-RTA sdAb C8 were transferred into the framework regions of sdAb A3 to produce the thermostabilized A3C8 sdAb.⁵⁹ Two pet22 constructs of A3C8 were prepared for periplasmic expression─ a C-terminal His-tagged plasmid and a tag-free construct (referred to as A3C8stop). The protein sequences are shown in the Suppl. Information.

Protein expression and purification of sdAb

The His-tagged sdAb were periplasmically expressed and purified using the methods previously described in George et al.³⁹

Protein expression and purification of RTA and RTA1-33/44-198 and its variants

RTA and RTA1-33/44-198 were purified as previously described.³⁰ Briefly,E.coli BL-21(DE3) were grown in LB media supplemented with Kanamycin (50 µg/ml) at 37°C. Cultures at a cell density of 0.8–1.0 OD₆₀₀ were induced by addition of IPTG (0.2 mM) and grown at 17°C for 18–20 hours before collection by centrifugation. Cell pellets were resuspended in lysis buffer (50 mM sodium phosphate buffer, pH 7.3, 2 mM EDTA, 30% BugBuster®) and sonicated, 30 seconds on/30 seconds off, for a total of 2 minutes. The lysate was clarified by centrifugation at 20,000 × g for 30 min at 4°C. The supernatant was applied to a Q Sepharose column equilibrated with 50 mM sodium phosphate buffer, 2 mM EDTA, pH 7.3, and the protein was collected in the flow through. Fractions containing the protein were combined and dialyzed overnight at 4°C against 1 L of 50 mM MES, 2 mM EDTA (pH 5.5 for RTA and pH 6.4 for RTA1-33/44-198). Dialyzed protein was loaded onto a SP Sepharose column equilibrated with 50 mM MES, 2 mM EDTA pH 5.5 for RTA and pH 6.4 for RTA1-33/44-198. Protein was eluted from the column using a 0–250 mM sodium chloride gradient and analyzed using SDS-PAGE. The fractions containing a single band on SDS-PAGE gels were combined, dialyzed into 50% glycerol, and stored at −20°C.

Preparation and purification of the RTA:sdAb complexes

RTA:sdAb complexes were prepared by incubating RTA or RTA1-33/44-198 with a 10% molar excess (1:1.1 molar ratio) of each sdAb at 4° C for >17 hours. The concentration of RTA or RTA1-33/44-198 in 0.5 mL was determined by UV-vis using calculated extinction coefficients, ϵ = 26,000 M⁻¹cm⁻¹ or ϵ = 18,005 M⁻¹cm⁻¹, respectively. The calculated extinction coefficients for each sdAb were the following: 36,565 M⁻¹cm⁻¹ for C10neg; 23,045 for H1W12V and F6; and 21,555 M⁻¹cm⁻¹ for the remaining sdAbs. Each complex was applied to a Superdex G-75 or G-200 gel-filtration column (as appropriate) equilibrated in 1x phosphate-buffered saline (PBS) pH 4.5 or pH 7.4 to separate the formed complex from excess sdAb. To verify the formation of each complex, each column was calibrated using 3 molecular weight (MW) standards: albumin (66,000 Da); carbonic anhydrase (29,000 Da); and cytochrome C (12,400 Da). Calibration curves for each column at pH 4.5 or pH 7.4 were made using a log-linear plot of molecular weight (kDa) vs. elution volume (mL) and fitting the points to a single exponential decay equation using Grafit 5.0 (Erithricus Software Ltd. West Sussex, UK). The MWs of each complex were calculated from the elution volumes and the protein sequence for comparison and confirmation of complex purification.

Circular dichroism and thermal denaturation curves

Thermal denaturation (2°C/min) of RTA, RTA1-33/44-198, the sdAbs, or their complexes, was monitored using a Jasco 810 Circular Dichroism (CD) spectropolarimeter fitted with a Peltier temperature controller. Concentrations of protein solutions (˜0.2 mg/ml in PBS, pH 4.5 or pH 7.4) were determined by UV-vis using the appropriate calculated extinction coefficient. Melting curves were measured between 10–95°C by monitoring the change in ellipticity at 207, 214, or 222 nm. The apparent melting temperature (T_m) was determined from a 4 parameter fit of data. The three T_m values were averaged and standard deviations (SD) were calculated.

Western analysis of site-directed mutants

Proteins were first separated by SDS-PAGE using an 8–16% gradient gel. Equal amounts of RTA, RTA1-33/44-198, or their variants were loaded (5 μg/lane). RTA isolated from ricin (glycosylated) was used as a control. The proteins in the gel were transferred to nitrocellulose and blocked for 1 h using 1x TBST containing 5% (w/v) dry milk. The primary Ab was incubated in 1x TBST and 5% dry milk for 17 hours at 4°C. The blot was washed 3 times with 1x TBST between subsequent Ab incubations (1 h incubation at 22 ± 3°C) and then developed. SdAb A3C8 containing a C-terminal His-tag was used as the primary Ab (0.1 mg/mL). A mouse anti-His-tag antibody was used as the secondary Ab (1:500 dilution). An anti-mouse AP- conjugated IgG (1:500 dilution) and stabilized AP substrate were used for detection as directed.

Cell-free translation assay

Ricin biological activity was determined using a microtiter cell-free translation assay that detects luciferase translation from luciferase mRNA.⁶⁰ Dilutions of ricin are included in each assay for generation of a standard curve. Briefly, antibodies were diluted with PBS in 96-well microtiter plates. A constant amount of ricin (100 ng/mL final concentration) was added to the Ab dilutions, the plate left on a microplate shaker for 15 min (25° C), and then 5 μL was transferred to a v-shaped microtiter plate. The rabbit reticulocyte lysate, RNasin, amino acids complete, and luciferase mRNA were mixed together, and then 25 μL was added to each well. The plates were incubated (37°C) for 90 min, and then 5 μL of the translation lysate was transferred to a black microtiter plate. After the addition of the assay buffer, containing the luciferin substrate, luminescence was measured as relative light units (RLU) using a SpectraMax M5 Multi-Mode Microplate reader (Molecular Devices, LLC., Sunnyvale, CA). The average ± SD of 3 wells was reported as the percentage (%) untreated control, [(treated/untreated control) × 100].

In vitro cell cytotoxicity assay

The mouse lymphoma EL-4 cell line⁶¹ (ATCC, Manassas, VA) was maintained in RPMI-1640 medium supplemented with 5% fetal calf serum. Immediately prior to the assay, cells were pelleted (600 × g, 10 min, 4° C) and resuspended to 2 × 10⁶ cells/mL in RPMI-1640 medium. Using RPMI-1640 medium, sdAbs were diluted (50 μL/well) in a 96-well microtiter plate. Ricin was diluted to yield a final concentration of 200 ng/mL and was added (50 μL/well) to the antibody dilutions. After the plates had been left on a shaker for 15 min, cells (100 μL) were added to each well. The plates were incubated (37° C, 5% CO₂) overnight (˜17 h) after which 20 μL Cell Titer 96 was added to each well. The plate incubated for 4 additional hours. Dilutions of ricin were included in each assay and used to generate a standard curve. Optical density (490 nm) was measured on a Microplate reader. The average ± SD of 3 wells was reported as percentage (%) untreated control cells, [(OD of treated cells/OD untreated control cells) × 100].

Crystallization

SdAb A3C8 with a C-terminal His-tag (A3C8cterm) was crystallized in the ammonium sulfate grid screen condition B2 (0.1 M citric acid pH 5.0, 1.6 M ammonium sulfate) at 17°C. Protein in 50 mM Tris pH 7.6 was concentrated to 3.5 mg/mL. Hexagonal plates were obtained in hanging drops containing 1:1 mixtures of protein and precipitant.

RTA1-33/44-198 and A3C8cterm were mixed at a 1:1.1 molar ratio and was exchanged into 50 mM Tris pH 7.6, concentrated (12.5 mg/mL) and crystallized in the ammonium sulfate grid screen condition B4 (0.1 M HEPES pH 7.0, 1.6 M ammonium sulfate). Crystals grew at 17°C within a month.

X-ray crystallography

A3C8 crystals were cryo-protected in 30% glycerol, 70% ammonium sulfate grid screen condition B2. Crystals of the RTA1-33/44-198:A3C8cterm complex were cryo-protected in 30% glycerol and 70% precipitant (0.1 M HEPES pH 7.0, 1.6 M ammonium sulfate). The RTA1-33/44-198:A3C8 crystals contained 4 molecules (2 sdAb, and 2 RTA or RTA1-33/44-198 variants) per asymmetric unit.

Data were collected at 150 K using a Bruker Micro-STAR rotating anode equipped with Helios optics and a Bruker Platinum 135 CCD area detector. The structure of the complex was solved by molecular replacement using Phaser.⁶² The structures of RTA (PDB 1J1M),³⁸ RTA1-33/44-198 (PDB 4IMV),²¹ a sdAb (PDB 4FHB),⁵¹ and the structure of the A3C8 sdAb (PDB 5SV4) were used as search models. Model-building was performed using Coot.^63,64 Simulated annealing was carried out using CNS 1.1,⁶⁵ and refinement with Refmac 5.^66,67

Surface plasmon resonance of site-directed mutants of RTA

The dissociation constants, K_d, for each of the Abs was determined using a ProteOn™ XPR36 Protein Interaction Array System (Bio-Rad) as described previously.⁶⁸ All tests were performed on a GLC sensor chip, for general amine coupling and with a binding capacity of approximately a one protein monolayer. Following activation of the sensor chip with 0.1 M 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and 0.25 M N-hydroxysulfosuccinimide, immobilization was performed in 10 mM acetate buffer pH 5.0 for immobilization of RTA or its variants at 20 µg/mL on all 6 lanes. The binding kinetics of each sdAb were determined by rotating the chip and flowing various concentrations (300, 100, 33, 11, 3.7, 0 nM) over the chip at 100 µL/minute in the orthogonal direction for 90 s over the RTA-coated chip (or RTA variants) and then monitoring dissociation for 600 s using PBS with 0.005% Tween-20 (pH 7.4) or PBS with 13 mM sodium citrate (pH 4.5) as the running buffer. The surface was regenerated at 50 µL/minute with 1% phosphoric acid for 36 s. Data were corrected by subtraction of the zero antigen concentration column as well as by interspot correction.

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