Small-Molecule Probes for Affinity-Guided Introduction of Biocom

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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

Small-Molecule Probes for Affinity-Guided Introduction of Biocompatible Handles on Metal-Binding Proteins Michael R. Mortensen,†,‡ Mikkel B. Skovsgaard,†,‡ Anders H. Okholm,† Carsten Scavenius,§ Daniel M. Dupont,† Christian B. Rosen,†,‡ Jan J. Enghild,§ Jørgen Kjems,† and Kurt V. Gothelf*,†,‡ †

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Center for Multifunctional Biomolecular Drug Design at the Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 C, Aarhus, Denmark ‡ Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 C Aarhus, Denmark § Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, 8000 C Aarhus, Denmark S Supporting Information *

ABSTRACT: Protein conjugates of high heterogeneity may contain species with significantly different biological properties, and as a consequence, the focus on methods for production of conjugates of higher quality has increased. Here, we demonstrate an efficient and generic approach for the modification of metal-binding proteins with biocompatible chemical handles without the need for genetic modifications. Affinity-guided small-molecule probes are developed for direct conjugation to off-the-shelf proteins and for installing different chemical handles on the protein surface. While purification of protein conjugates obtained by small molecule conjugation is troublesome, the affinity motifs of the probes presented here allow for purification of the conjugates. The versatility of the probes is demonstrated by conjugation to several His-tagged and natural metal-binding proteins, including the efficient and area-selective conjugation to three therapeutically relevant antibodies.

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INTRODUCTION Protein conjugates have found wide use in sensing techniques, imaging techniques, and the drug-delivery field, which have spurred the development of several methods for their construction. Random labeling approaches such as NHSester labeling produce complicated product mixtures without control of labeling position, while affinity-guided approaches have shown a higher degree of site-selectivity.1−3 In affinityguided protein conjugation, probes are designed to form a complex with the protein of interest. The complex increases the local concentration of the reactive group of the probe at the protein surface, facilitating the conjugation reaction with the protein. Since the complex restricts the reach of the probe, affinity-guided conjugations are generally selective for an area in the vicinity of the complexation site. Ligands,4,5 boronic acids,6 and metal chelators7 have been utilized for complex © XXXX American Chemical Society

formation. Metal chelator-guided probes have generally been limited to the labeling of His-tagged proteins,7−10 thus, limiting the applicability to recombinant proteins. We have previously shown that metal complexes can be used to guide protein labeling of inherent metal-binding proteins, but the labels were limited to oligonucleotides3 or the protein scope limited to nonglycosylated proteins.11 A recent approach for labeling of metal-binding proteins used palladium for the conjugation but required elevated reaction temperatures and inactivated the protein of interest.12 Here, we have expanded the toolbox of affinity-guided small molecules by the design and synthesis of two complex probes Received: June 16, 2018 Revised: August 8, 2018 Published: August 9, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00424 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Metal-mediated conjugation to proteins forming site-selectively modified conjugates. (a) Illustration of metal affinity-guided protein conjugation (PDB entry 3QYT). The metal-binding moiety of the probe forms a metal complex with the protein metal-binding site facilitating a reductive amination reaction between the aldehyde of the probe and a lysine residue in proximity of the metal binding site on the protein surface. (b) The reaction paths of the probes for the labeling of metal-binding proteins. First, the probes are conjugated to the protein in a metal complex guided reaction. The cleavable linker is cleaved revealing reactive handles for further modification. Lastly, the reactive handles are modified in chemoselective reactions. (c) Metal dependent labeling of His6-GFP, serotransferrin, and anti c-Myc with Azide-Probe 1 followed by SPAAC with DBCO-Cy5.

that integrate all function (affinity and purification motifs, protein conjugation group, and biocompatible handle) in one

molecule. The two probes enable efficient affinity-guided introduction of either an azide or an aniline handle on metal B


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jugated with the Azide-Probe 1 and labeled with a Cy5 fluorophore by SPAAC. The binding of the fluorescent serotransferrin conjugates to a transferrin receptor positive cell line was investigated by confocal microscopy and flow cytometry (Figure S3). Incubation with the fluorescent conjugates produced a significant increase in fluorescence compared to untreated cells, indicating binding to the cells. Competition experiments with 10× excess of unmodified serotransferrin to conjugate resulted in a decrease in fluorescence, indicating retained selectivity for the binding of the transferrin receptor (Figure S3). We confirmed an areaselective labeling of serotransferrin by tandem MS of tryptic digests of purified single-conjugated aniline-modified protein. As expected, the analysis suggests that the modifications are positioned at lysines in close proximity to the metal-binding site (Figure S4). From the labeling of an inherent metalbinding protein with a dedicated metal-binding pocket, we turned to the labeling of inherent metal-coordinating proteins that chelates the metal through surface exposed residues. Optimizing Conditions for Antibody Conjugation. Before investigating the probes for conjugation to a series of therapeutically relevant antibodies, the conjugation conditions were optimized for the therapeutic antibody trastuzumab (Herceptin). Trastuzumab is part of the IgG1 antibody subclass that contains a cluster of 3−4 histidines on the Fcdomain, which is sufficient for templating the conjugation reaction.3 In accordance with the Irving-Williams series, we find that Cu2+ is the most efficient divalent cation for guiding of the conjugation reaction (Figure S5).15 The reactions are generally performed overnight at room temperature; however, increased conversion is observed for the Azide-Probe 1 at longer reaction times (Figure S6). The conjugation efficiency is independent of salt concentrations between 100 and 400 mM (NaCl or Na2SO4, Figure S7), thus, the conjugations are generally performed with 100 mM NaCl. Neutral pH (7.5) is found to be the optimal for the conjugation (Figure S8), indicating that increased stability of the metal complex increases the conjugation efficiency, since the NTA-metal complex is stabilized at higher pH. The reaction is performed at low concentrations of protein (2 μM) and probe (2−25 equiv) to avoid nondirected side reactions. Surprisingly, the efficiency of the conjugation is independent of concentration between 2 and 8 μM of the protein (Figure S9). Furthermore, 25 equiv of the probe can be utilized without interference of significant nondirected side reaction (Figure S10). We attribute the ability of using higher concentrations of both protein and probe without significant observation of nondirected reaction to the relatively slow kinetics of the nondirected reductive amination reaction. Modification of Therapeutic Antibodies. Several antibodies have been approved as cancer therapeutics,16 including the IgG1 antibodies, rituximab (MabThera), cetuximab (Erbitux), and the antibody mentioned above, trastuzumab (Herceptin) . We have investigated the conjugation to these therapeutically relevant antibodies because they have shown promising results as drug carriers (Figure 2a).17−19 The antibodies are purchased as the drug formulations, and we demonstrate that they can be labeled in a metal guided conjugation reaction using the Azide-Probe 1 followed by a SPAAC reaction with DBCO-Cy5 dye (Figure 2b). The conjugation to the antibodies is further analyzed by anion exchange chromatography using different equivalents of both the Azide- and Azo-Probes (Figures S11−S13). One of the

binding proteins in a chemo- and area-selective manner without the need for genetically engineered proteins. As a further asset, we demonstrate that the affinity groups of the probes can be used for purification of the conjugates, which is generally challenging for conjugates formed by small molecules. For one of the probes, the affinity groups can be quantitatively removed revealing the small aniline modification as a reactive scar for further modification. The conjugation could be performed on a variety of metal-binding proteins including therapeutic IgG1 antibodies.

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RESULTS AND DISCUSSION Probe Design and Synthesis. We developed metal complex guided probes to allow for conjugation to a wide variety of nongenetically engineered proteins, since an estimated 25−50% of all proteins are capable of coordinating metal ions.13 Metal complexation between the probe and protein increases the local concentration of the probes reactive group at the surface of the protein, facilitating the conjugation reaction that installs a biocompatible handle on the protein (Figure 1a). Upon removal of the coordinating metal from the conjugates with EDTA, the metal coordinating groups would contain several charges that we believed could be utilized for purification of the conjugates, which is generally extremely challenging for small molecule−protein conjugates. We designed the probes with two nitrilotriacetic acid (NTA) metal-coordinating groups for metal complexation with metalbinding sites of the proteins, an aldehyde for conjugation by reductive amination, and a linker region carrying the azide (Azide-Probe 1) or concealing the aniline handles (Azo-Probe 2). The azide was inserted in the probe with a branched noncleavable linker and can be selectively modified by a strain promoted azide alkyne cycloaddition (SPAAC) when conjugated to the protein (Figure 1b). The aniline functionality was masked with a cleavable linker that upon cleavage simultaneously liberates the aniline functional scar and removes the metal binding moieties from the protein conjugate (Figure 1b), and thus only the small aniline modification remains on the protein. Furthermore, the aniline functionality can be used in a biocompatible oxidative coupling reaction developed by Francis and co-workers.14 The probes were synthesized in a modular fashion (Figure S1), which allows for the rapid exchange of electrophiles, linkers, and affinity guiding moieties. Versatility of the Bioconjugation. Initially, we investigated the versatility of the conjugation reaction by evaluating the labeling of several metal-binding proteins with different metal coordination geometries. Both His-tagged (His6-GFP and IL6-His6) and inherent metal binding proteins (iron transport protein serotransferrin, antibody anti c-Myc, metalloenzymes carboxypeptidase, and carbonic anhydrase) demonstrated metal complex dependent labeling with the AzideProbe 1 followed by SPAAC reaction with DBCO-Cy5 for visualization of the protein conjugates (Figures 1c and S2). The preorganization provided by the metal complex is crucial for the efficiency of the conjugation reaction, which becomes evident when the divalent metal ion is omitted. In addition, the reactant concentrations of affinity-guided reactions are kept low to reduce nonguided conjugation that would lack the desired area-selectivity. Serotransferrin Conjugates. We further investigated the impact of the labeling on the inherent metal-binding irontransporting protein serotransferrin. The protein was conC


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Figure 2. Conjugation to therapeutic IgG antibodies. (a) The reaction chemistry for both the Azide-Probe 1 and the Azo-Probe 2. The conjugation reaction is performed using the Azide-Probe 1 (upper path) followed by a strain-promoted click reaction to attach the desired label. If the conjugation reaction is performed using the Azo-Probe 2 (lower path), the linker can be reductively cleaved revealing an aniline moiety. The aniline can be selectively coupled to the label through an oxidative labeling reaction. The antibody crystal structure was adapted from PDB: 1IGT. (b) SDS-PAGE analysis of the conjugation and labeling of the therapeutic antibodies (trastuzumab: Tz, rituximab: Rx, cetuximab: Cx) using the AzideProbe 1 and DBCO-Cy5 (10 equiv). The same gel was scanned for Cy5 fluorescence (top) and stained for protein by SimpleBlue SafeStain (bottom). (c) Anion exchange chromatographic analysis of the conjugation to the therapeutic antibodies using the metal-guided probes. The conjugation is highly efficient, providing near quantitative conversion of the unmodified protein using the Azo-Probe. The anion exchange chromatograms are shown in Figures S11−S13. (d) Unmodified, single and double aniline-modified trastuzumab were conjugated with oaminophenol-PEG10K using K3Fe(CN)6 as an oxidant. The analysis of the band intensities is shown in Figure S25.

major assets of these probes is that the NTA groups enable separation of the unmodified, monolabeled, and dilabeled antibodies from the conjugation reactions. We confirmed the identities of the conjugates by MALDI-TOF-MS (Figures S14−S16). The chromatographic analysis revealed that the three therapeutic antibodies perform similarly in the affinityguided conjugation reaction. It also shows that the Azo-Probe 2 efficiently converts the unmodified antibodies to conjugate, and only 5 eq. of the probe is required for obtaining near full conversion of unmodified protein (Figure 2c). However, the Azide-Probe 1 is less efficient, which may originate from the branched structure of the probe causing steric repulsion between the linker arm containing the azide functionality and the protein. The kinetics of the labeling using the Azo- and Azide-Probes was compared using anion exchange chromatography (Figures S17−S20). The Azo-Probe 2 shows completion of the conjugation reaction after approximately 6 h, while the

Azide-Probe 1 requires longer reaction time (24 h) in accordance with our previous analysis. Having established an efficient protocol for conjugation and purification, we next investigated the conversion of the SPAAC reaction on single- and dual azide-modified trastuzumab (Figure S21). The reaction was performed with DBCOPEG20.000, yielding approximately 52−65% conversion, in accordance with the literature.20 To generate a functional handle and remove the metal affinity groups from proteins modified by the Azo-Probe 2, the azobenzene-linker is cleaved under mild conditions using the reductant Na2S2O4 to form an aniline.21 The cleavage conditions are investigated for trastuzumab using 10 mM reductant, revealing quantitative liberation of the resulting aniline after just 10 min (Figure S22). Since the NTA-moieties allow for purification of the conjugates by anion exchange chromatography, it is of paramount importance to achieve high conversion in the subsequent cleavage step in order to reduce D


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Figure 3. Investigating the site-selectivity of the antibody conjugation. (a) Schematic of the experimental steps performed prior to the analysis in b. The numbers correspond to the lane numbers in b. (b) SDS-PAGE analysis of the site-selectivity of the conjugation reaction on trastuzumab. The antibody and fragments were labeled according to the reaction sequences in a. The conjugation reaction of the full antibody shows high selectivity for the Fab domain (lane 1). However, when conjugation is performed on the Fab and Fc-fragments, the selectivity switches to the Fc-domain (lane 2), indicating the importance of the metal-binding histidine cluster. Lane 3 shows the unspecific coupling of the DBCO-Cy5 reagent. (c) Crystal structure of the Fab domain of trastuzumab. The sites of modifications for monolabeled trastuzumab found by tandem MS analysis of trypsin digests of the full antibody are shown in red. *Lys226 is positioned in the hinge region (close to the other modifications), which is not part of the crystal structures available for trastuzumab (PDB entry 1N8Z).

this experiment, the fluorescence appears mainly from the band corresponding to the Fc-domain (Tz: Figure 3b, lane 2. Rx, Cx: Figure S26). This confirms that it is mainly the metalbinding site positioned at the Fc-domain that directs the conjugation reaction. Lane 2 also shows that a small fraction of the Fab domain is labeled. This may be caused by unspecific reaction of the DBCO reagent or a weak directing effect from individual histidines at the surface of the Fab-domain. We believe the high flexibility of the hinge region in the antibodies allows the aldehyde of the probes to react at the Fab-domain in the intact antibody.22 To support these results, we analyzed trypsin digests of HPLC-purified and reductively cleaved azobenzene-linker conjugates containing either one or two modifications by tandem MS (Figure 3c and Figures S27−29). For trastuzumab, rituximab, and cetuximab, our analysis confirms conjugation to lysines at the Fab-domain. It also indicates that the conjugation sites are located at the constant part of the Fab-domain close to the hinge region. This conjugation site is ideal due to its significant distance from the antigen-binding site, which decreases the risk of altering the antigen-binding sites. Furthermore, the Fc-domain is crucial for the recycling of the IgG antibodies through the neonatal Fc-receptor,23 which is also located a significant distance from the modification site. Binding Analysis of the Antibody Conjugates. We used surface plasmon resonance (SPR) to compare the affinity of the antibody conjugates with their unmodified counterparts. Only the affinities of the conjugates containing a single azo- or azide-modification are assessed. Cetuximab conjugates are captured on the surface using a protein A-modified chip. The extracellular domain of the EGFR is used to assess the Kd values, which shows similar affinities of the conjugates and unmodified cetuximab (Figures 4a and S30−32). This shows that the conjugation chemistry has no effect on the affinity of these conjugates for their target.

the number of HPLC purifications needed to isolate the modifiable conjugate. The aniline moiety is selectively functionalized in an oxidative coupling with an o-aminophenol in the presence of potassium ferricyanide (K3[Fe(CN)6]).14 Here we show that aniline-modified trastuzumab, originating from reactions with 1−5 eq. of the Azo-Probe 2, is labeled with an o-aminophenol modified fluorophore (rhodamine B, RhoB) and PEG10,000 (Figures S23−24). The oxidative coupling is also performed for purified trastuzumab containing either one or two handles (Figure 2d and S25), resulting in a conversion of 36−41%. The conversion for the oxidative coupling is similar to previously reported reactions with aniline-modified antibody Fc-fragments.14 Site-Selectivity of the Antibody Conjugation. As the metal-binding sites of the antibodies are located at the Fcdomain, it is expected that the conjugation site is also positioned at the Fc-domain in proximity of the interaction site, which was observed for DNA-templated protein conjugation.3 However, in contrast to DNA-templated protein conjugation we observe equal degree of labeling at the light and heavy chains (Figure 2b), indicating that the conjugation is not directed toward the Fc-domain. Initially, we investigate the position of the conjugation site by enzymatic cleavage of the hinge region separating the Fc- and Fab-domains. The cleavage is performed using bead-immobilized papain for both the azide-labeled and unmodified antibodies, which are subsequently labeled using DBCO-Cy5. The analysis shows that the majority of the fluorescence is located at the Fabdomains (Tz: Figure 3b, lane 1. Rx, Cx: Figure S26). In order to investigate if the labeling is in fact directed by metal complex formation at the histidine cluster on the Fc-domain, the reaction is also performed in the opposite order where the antibody is enzymatic cleaved into the Fab- and Fc-fragments and then labeled in the metal-guided conjugation reaction. In E


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Figure 4. Performance of the conjugates and visualization of cell surface receptors. (a) SPR sensorgrams for unmodified, purified monolabeled azide-modified and azobenzene-modified cetuximab (Cx). The table shows the measured kassociation, kdissociation, and Kd values of the full experiments for Cx and trastuzumab (Tz). The analysis indicates little change in affinity of conjugates compared to unmodified antibody. Experiments were performed in triplicates and uncertainties correspond to the standard deviation. (b) Confocal fluorescence microscopy images of the binding of Cy5-labeled therapeutic antibodies to their targets on cell surfaces. The fluorescence of the Cy5-conjugates is shown in red, and the cell nuclei was stained with DAPI shown in blue. SK-BR-3 expressing HER2 was used for trastuzumab (Tz), Ramos cells expressing CD20 was used for rituximab (Rx), and MDA-MB-231 expressing EGFR was used for cetuximab (Cx). NIH 3T3 cells were used as the target-deficient cell line. Scale bars: 50 μm.

rituximab binding has previously been reported, indicating that the Kd of the antibody−antigen binding is in the low micromolar range.25 As other reports suggest binding in the low nanomolar range for the full antigen,26 we pursue to investigate if the conjugates are active by other means. To assess the activity of the therapeutic antibody conjugates further, we have studied their ability to interact with live human cancer cell lines. Fluorescent conjugates of trastuzumab, cetuximab, and rituximab are prepared using the AzideProbe 1 for conjugation followed by labeling with DBCO-Cy5. The conjugates are incubated with cells expressing the targets for the antibodies HER2, EGFR, and CD20, respectively. Imaging by confocal microscopy results in a significant increase in fluorescence of the cells incubated with the conjugates, compared to the control cells (Figure 4b and Figure S36), showing that the conjugates are binding to their targets. Competition experiments containing 10× excess of the unmodified antibody relative to the conjugate show a markedly

The trastuzumab antibodies are not compatible with the protein A-modified chips, presumably due to binding of protein A to the Fab-domain,24 which inactivates the antigenbinding domain. Therefore, these antibodies and conjugates are immobilized using an NHS-ester activated chip. The extracellular domain of HER2 is used to measure the affinity of the conjugates compared to unmodified trastuzumab. Small differences in the affinity are observed (Figures 4a and S33− 35), but more importantly the conjugates retained subnanomolar affinity, thus, showing highly preserved binding affinity. It was attempted to analyze the affinities of the rituximab conjugates by SPR using both protein A- and NHSimmobilization of the antibodies. However, none of the setups indicate binding to an epitope-peptide fused to a thioredoxintag for both conjugates and unmodified rituximab. ELISA has also been attempted, but these experiments also fail to show binding to the epitope-peptide. To our knowledge, only a single SPR experiment that used a similar peptide to show F


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The small molecule probe (2−20 μM, 1−10 eq., typically 5 eq. was used) was added to a mixture of CuSO4 (4−40 μM, 2− 20 eq., 2 eq. compared to the probe) and HEPES-buffer (50 mM, pH 7.5) containing NaCl (100 mM). This mixture was allowed to stand for 5 min after which the protein of interest (2 μM) and NaBH3CN (50 mM) were added. The reaction was incubated at rt overnight. To the reaction mixture was added EDTA (2.5 mM), which was left for 30 min. The mixture was concentrated and washed twice with an appropriate buffer in an Amicon Ultra centrifugal filter (MWCO 10−100 kDa depending on the protein, 14100 g for 15 min pr. wash and 40 min last spin). [Tris-buffer (20 mM, pH 8.5) was used for HPLC or direct MALDI-TOF analysis. Cleavage buffer (azobenzene: 50 mM phosphate buffer, pH 6.5) was used, when a cleavable linker was to be cleaved as the next step. PBS (1X) was used for subsequent labeling of azide-modified conjugates.] HPLC-Purification. Conjugates were purified using anion exchange chromatography on an YMC BioPro QA column (100 × 4.6, 5 μM) using a Thermo Scientific Dionex UltiMate 3000 UHPLC+. Buffer A contained Tris-buffer (20 mM, pH 8.5) and buffer B contained Tris-buffer (20 mM, pH 8.5) with NaCl (500 mM) or KCl (500 mM). The purification was performed at a flow rate of 0.5 mL/min with a gradient of 0− 90% B over 17 min. The fractions containing the conjugates were collected and concentrated in an Amicon Ultra centrifugal filter (MWCO 3− 100 kDa depending on the size of the protein, 14100 g for 10− 30 min). The concentration was determined using absorbance at 280 nm measured on a ND-1000 NanoDrop spectrophotometer. MALDI-TOF Analysis of Proteins and Conjugates. The proteins were prepared for MALDI-TOF analysis by washing in an Amicon Ultra centrifugal filter (MWCO 3−100 kDa depending on the protein, 14100 g for 40 min) with either Tris-buffer (20 mM, pH 8.0) or Milli-Q-water. The protein solution (1 μL) was mixed with a 2% TFA in Milli-Q-water solution (1 μL) and DHAP-matrix (1 μL).29 The mixture was mixed and spotted on the MALDI plate. Labeling of the Azide-Modified Conjugates with DBCO-Reagents. To the azide-modified conjugates in PBS was added the DBCO-reagent (10 equiv). The reaction mixture was incubated at rt for 2 h to overnight. The excess reagent was removed using an Amicon Ultra centrifugal filter (MWCO 10−30 kDa, 14100 g for 40 min), or the reaction mixture was directly subjected to analysis by SDS-PAGE (NuPAGE 4−12% Bis-Tris). Cleavage of the Azobenzene-Linker and Subsequent Labeling with an o-Aminophenol Compound. To the protein containing solution (20−25 μL) was added 10−25 μL freshly prepared Na2S2O4 stock solution (50 mM in 50 mM phosphate, pH 6.5) − final concentration: 12.5 or 25 mM. The reaction mixture was mixed and incubated for 10 or 1 min, respectively. The excess Na2S2O4 was removed using an illustra MicroSpin G-25 column (GE Healthcare, prepared with 50 mM pH 6.5 phosphate buffer according to the manufacturer’s instructions). Remaining Na2S2O4 was removed by repeated washes in an Amicon Ultra centrifugal filter (MWCO 30 kDa, 14100 g for 10−30 min). The coupling conditions between the aniline and oaminophenol were described by Francis and co-workers.14 Briefly, to the protein (2−20 μM), in a phosphate buffer (50 mM, pH 6.5), was added the o-aminophenol compound (20−

decrease in fluorescence (Figure 4b and Figure S36), suggesting that the selectivity of the conjugates for the native target is retained. This finding is further established in a negative control experiment with cells that do not express the molecular targets for trastuzumab, rituximab, or cetuximab (Figure 4b bottom and Figure S36). The binding of conjugates to the respective cell lines is also characterized quantitatively by flow cytometry (Figure S37). These experiments confirm the retained functionality and high selectivity observed by confocal microscopy. Finally, the functionality of the conjugates prepared by the Azo-Probe 2 is also tested. Fluorescent conjugates of the therapeutic antibodies, prepared by conjugation with the AzoProbe 2, reductive cleavage, and labeling with o-aminophenolRhoB, are incubated with cells containing the respective targets for the antibodies. Fluorescence microscopy images show binding of the conjugates to their targets on cell surfaces (Figure S38), and competition experiments with excess unmodified antibody suggested that the binding is selective for the natural target. Therefore, we conclude that these conjugates behave similarly to the Cy5-modified conjugates prepared with the Azide-Probe 1.

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CONCLUSION In summary, we have developed efficient probes for area- and chemoselective conjugation to native metal-binding and Histagged proteins. The probes install a biocompatible handle, azide or aniline, on the protein surface, which can be further modified in SPAAC and oxidative coupling reactions, respectively. The probes rely on an aldehyde for conjugation by reductive amination that conserves the charge of the modified lysine residue, which is important to avoid increased whole body clearance and aggregation of antibody conjugates.27,28 In addition, the metal affinity groups allow for purification of the conjugates, which is otherwise very challenging for small molecule−protein conjugates. Furthermore, the affinity groups can be efficiently removed leaving only the small aniline modification, when labeling with the Azo-Probe. Investigation of the conjugates indicated that the proteins remained functional, and for antibody conjugates the binding selectivity and affinity was highly preserved. We are currently pursuing improvements of the areaselectivity of the probes to achieve site-specific labeling by changing and tuning the electrophile. Since the small molecule probes have altered area-selectivity compared to DNAtemplated protein conjugation, we are investigating the possibility of predetermining the labeling site of affinity probes, overcoming the limitations of probes relying on differences in residue-microenvironment.

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EXPERIMENTAL PROCEDURES The protocols for the synthesis of the probes are described in the Supporting Information. All concentrations presented in this section are final concentrations unless otherwise noted. General Procedure for the Protein Labeling Reaction Using the Small Molecule Probes. The reactions were performed on scales ranging from 20 to 1000 pmol. Generally, final concentrations 2−6 μM of proteins were used. The equivalents have been calculated according to the protein content. G


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subsequently washed three times in PBS and fixed and stained according to the standard protocol. For flow cytometry, 200,000 cells/well in a 24 well plate were treated with 100 nM cetuximab-Cy5 in fresh growth medium for 1 h at 37 °C. For the competition experiment, cells were charged with 1 μM cetuximab 5 min before incubation. After treatment cells were washed three times in PBS and then detached from the well bottom using a cell scraper. Cells were recovered in 0.5 mL of PBS, vortexed, and analyzed by flow cytometry. Trastuzumab. For experiments with trastuzumab, the same procedure as used for cetuximab was followed, with the exception that instead of MDA-MB-231 cells, SK-BR-3 cells expressing the human epidermal growth factor receptor 2 were used. Microscopy. Microscopy was performed on a confocal laser scanning microscope (LSM 700; Zeiss, Jena, Germany) equipped with solid state lasers delivering light at 405, 488, 555, and 633 nm, respectively. Light was collected through a 20×/0.8 objective and a 63×/1.4 Oil DIC immersion objective (Zeiss). DAPI fluorescence was excited with the 405 nm line, Alexa Fluor 488 conjugate of WGA was excited with the 488 nm line, while Cy5 fluorescence was excited using the 633 nm line. RhoB was excited with the 555 nm line. Digital image recording was performed with the ZEN 2012 software (Zeiss), and image analysis was performed using Fiji (ImageJ). Flow Cytometry. Flow cytometric analysis was performed on a Gallios Flow Cytometer (Beckman Coulter) using the 638 nm laser. A maximum of 10,000 cells were assessed in each experiment. Forward and side scatter was used to gate a homogeneous population of single cells. FCS files were analyzed using Kaluza. Tandem MS. Tryptic Digest. The samples were denatured, reduced, and alkylated in 20 mM Tris-HCl, 6 M urea, pH 8 containing 5 mM DTT followed by the addition of iodoacetamide to a final concentration of 15 mM. The reduced and alkylated samples were diluted 5 times with 20 mM NH4HCO3 and digested with trypsin (1:25 w/w) at 37 °C for 16 h. The tryptic peptides were micropurified using Empore SPE Disks of C18 octadecyl packed in 10 μL pipet tips.30 Mass Spectrometry. NanoESI-MS/MS analyses were performed on an eksigent nanoLC 415 system (Sciex) connected to a TripleTOF 6600 mass spectrometer (Sciex) equipped with a NanoSpray III source (AB Sciex). The micropurified peptides were suspended in 0.1% formic acid and injected and trapped isocratically on an in-house packed trap column (2 cm × 100 μm, RP ReproSil-Pur C18-AQ 3 μm resin, Dr. Maisch GmbH). The peptides were eluted from the trap column and separated on a 15 cm analytical column (75 μm i.d.) packed in-house in a pulled emitter with RP ReproSilPur C18-AQ 3 μm resin (Dr. Marisch GmbH, AmmerbuchEntringen, Germany). Peptides were eluted using a flow of 250 nL/min and a 20 min gradient from 5% to 35% phase B (0.1% formic acid and 90% acetonitrile). The collected MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.1 (AB SCIEX) and the “proteinpilot MGF” parameters. The generated peak lists were searched against a custom database containing the modified proteins using an in-house Mascot 2.5.1 search engine (matrix science). Search parameters were allowing one missed trypsin cleavage site and carbamidomethyl as a fixed modification with peptide tolerance and MS/MS tolerance set to 15 ppm and 0.2

200 μM, 10 equiv) and K3Fe(CN)6 (200−2000 μM, 100 equiv). The reaction was performed over 25 min at rt. The reaction mixture was diluted with phosphate buffer, and excess reagents were removed by repeated washes in an Amicon Ultra centrifugal filter (MWCO 10−30 kDa, 14100g for 40 min). Study of Site-Selectivity Using Hinge Region Cleavage. The fluorescent conjugates were formed using the general labeling protocol and cleaved in the hinge region using a Pierce Fab preparation kit (Thermo Scientific) according to the manufacturer’s protocol. Cell Culture. KB-3-1 and Ramos cells were maintained in full RPMI-1640 Glutamax medium (Sigma), MDA-MB-231 cells and NIH 3T3 cells were maintained in DMEM AQmedia (Sigma), and SK-BR-3 cells were maintained in DMEM (Sigma). All cell cultures were supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (50 μg/mL) and kept at 37 °C in humidified air containing 5% CO2. Transferrin Uptake Experiments. 40,000 KB-3-1 cells/ well were seeded in 8-well chamber slides (Sarstedt) and grown overnight. On the next day, the medium was exchanged by washing the cells with FBS-free RPMI-1640 medium. Prior to uptake experiments, Apo-Tf was loaded with Fe(III) by incubation with 10 molar excess of FeCl3 in 100 mM NaHCO3, pH 8.0, for 2 h. Cells were incubated with ironsaturated (holo-) Tf-Cy5 at a final concentration of 100 nM. After 15 min incubation, the cells were washed three times with PBS. To assess the specificity of uptake, competitive binding assays were performed by pretreating the cells with 1 μM native holo-Tf 5 min before addition of holo-Tf-Cy5. For microscopy, cells were fixed by incubation with 4% PFA in PBS for 15 min at room temperature followed by three PBS washes. The membranes were stained in 5 μg/mL Alexa Fluor 488 conjugate of WGA (Invitrogen) for 10 min at room temperature and washed in PBS twice. The cell slide was dried under a stream of nitrogen and mounted using ProLong Gold antifade mauntant with DAPI (Thermo Fischer). The mounting media was allowed to settle for 24 h before microscopy. Immunofluorescence Experiment. All experiments with anticancer antibodies were repeated with NIH 3T3 mouse fibroblasts as negative control cells. Rituximab. For a typical experiment, 600,000 Ramos cells expressing the CD20 antigen were incubated in growth medium with 100 nM rituximab-Cy5 for 1 h at 37 °C. For the competition experiment, cells were charged with 1 μM unmodified rituximab 5 min before incubation. Cells were washed three times in PBS by centrifugation at 300 × g for 5 min. 200000 cells were then analyzed by flow cytometry. To enable microscopy, 400,000 cells in 50 μL of PBS were incubated in 8-well chamber slides for 30 min at 37 °C. Prior to incubation, cell chambers were treated with 0.05 mg/mL poly-L-lysine (>30 000 Da, Sigma) for 10 min at 37 °C and washed twice with PBS. Cells were subsequently fixed and stained according to the protocol used for transferrin uptake experiments. Cetuximab. For microscopy analysis, 40,000 MDA-MB-231 cells expressing the epidermal growth factor receptor were seeded in 8-well chamber slides overnight in growth medium. On the next day, the cells were treated with 100 nM cetuximab-Cy5 in fresh growth medium for 1 h at 37 °C. For the competition experiment, cells were charged with 1 μM unmodified cetuximab 5 min before incubation. Cells were H


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(WO2016005474 A8), which in part covers the technology. The other authors declare no competing financial interests.

Da, respectively. To identify the site of modification, the Azomod (full or hydrolyzed) was set as variable modifications of lysine residues. All sites of Azo-modification were manually inspected and validated. Spectra with modified peptides were extracted by importing all data with an ion score cutoff of 30 and a significant threshold of 0.01 (p < 0.01) into MS Data Miner v. 1.3.0 (MDM).31 From MDM all modified peptides were exported. Surface Plasmon Resonance Protocol. SPR analysis of antigen−antibody interactions was performed on a Biacore T200 instrument (GE Healthcare) at 25 °C using different setups. All binding reactions were conducted in 20 mM HEPES (pH 7.4), 140 mM NaCl, 0.005% Tween20 using a flow rate of 30 μL/min. Sensor chips were purchased from XanTec bioanalytics GmbH. The interaction of cetuximab antibodies with EGFR ECD (EGR-H82E0-25 μg from Acrobiosystems) was measured using protein A sensor chips (PAD-50L). Antibody (5−10 nM) was captured on one flow cell to a level of 150−200 RUs. On the reference flow cell no antibody was captured. Then, 2fold dilution series (10 nM−0.625 nM) were injected for 180 s (association), and the dissociation followed for 300 s. The sensor surface was regenerated with a 30 s injection of 10 mM glycine-HCl (pH 2.5) after each run. The interaction of trastuzumab antibodies with HER2 ECD (HE2-H822R from Acrobiosystems) was measured with CMD50L sensor chips. The trastuzumab antibodies (15−25 μg/mL antibody in 10 mM NaOAc pH 5) were individually immobilized on the flow cells to a level of ∼350 RUs. The reference flow cell was subjected to a blank immobilization. HER2 ECD association (120 s) and dissociation (1200 s) were measured upon injections of 2-fold dilution series (50 nM− 3.125 nM). Between cycles, the sensor surface was regenerated by the injection of 10 mM glycine-HCl (pH 2.5) for 45 s. All binding reactions were analyzed by the kinetic software in the Biacore T200 evaluation program, based on a 1:1 stoichiometry, and KD values estimated from the association and dissociation rate constants.

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ACKNOWLEDGMENTS We want to thank Assistant Professor Thomas Tørring and Dr. Anne Louise B. Kodal for discussions in relation to the project. Furthermore, we would like to thank Claus Bus for maintenance of the cell lines and Dr. Line Hansen for assistance with the Ramos cell experiments. The work was funded by the Danish National Research Foundation (grant number DNRF81) and Faculty of Science and Technology, Aarhus University. Furthermore, we would like to thank the Lundbeck Foundation (grant number R180-2014-3127) for support to the Ph.D. of M.B.S.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00424. General materials and equipment information; supporting figures of reaction optimization, confocal laser microscopy images of fluorescent conjugates, flow cytometry analysis of fluorescent conjugates, tandem MS analysis, MS of conjugates, SPR sensorgrams of conjugates; synthesis and analysis of small molecule probes (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Michael R. Mortensen: 0000-0003-0261-1385 Kurt V. Gothelf: 0000-0003-2399-3757 Notes

The authors declare the following competing financial interest(s): M.R.M., C.B.R. and K.V.G. are authors of a patent I


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