Small-Molecule Probes for Affinity-Guided Introduction of

Subscriber access provided by AUSTRALIAN NATIONAL UNIV

Article

Small-Molecule Probes for Affinity-Guided Introduction of Biocom-patible Handles on Metal-Binding Proteins Michael Rosholm Mortensen, Mikkel Skovsgaard, Anders H Okholm, Carsten Scavenius, Daniel M Dupont, Christian Bech Rosen, Jan J Enghild, Jørgen Kjems, and Kurt Vesterager Gothelf Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00424 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

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* Center for Multifunctional Biomolecular Drug Design at the Interdisciplinary Nanoscience Center, Gustav Wieds Vej 14, 8000, Aarhus C and Denmark and Department of Chemistry, Langelandsgade 140, 8000 Aarhus C, Denmark. 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-motives 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.

INTRODUCTION Protein conjugates have found wide use in sensing techniques, imaging techniques and the drug-delivery delivery field, which have spurred the development of several methods for their construction. Random labeling approaches such as NHS-ester labeling produces complicated product mixtures without control of labeling position, while affinity guided approaches have shown a higher degree of site-selectivity.1–3 In affinity-guided 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 vicinity of the complexation site. Ligands,4,5 boronic acids6 and metal chelators7 have been utilized for complex 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 non-glycosylated 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 that integrate all function (affinity and purifi-

cation motifs, protein conjugation group and biocompatible handle) in one molecule. The two probes enables efficient affinity-guided introduction of either an azide or an aniline handle on metal 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.

RESULTS AND DISCUSSION Probe design and synthesis. We developed metal complex guided probes to allow for conjugation to a wide variety of non-genetically 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 metal-binding sites of the proteins, an aldehyde for conjugation by reductive amination

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 11

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. Firstly, 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.

2 ACS Paragon Plus Environment

Page 3 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 non-cleavable linker and can be selectively modified by strain-promoted azide alkyne click (SPAAC) chemistry 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 coworkers.14 The probes were synthesized in a modular fashion (Figure S1), which allows for the rapid exchange of electrophiles, linkers and affinity guiding moieties.

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 Fc-domain, 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 100-400 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 eq.) to avoid non-directed side-reactions. Surprisingly, the efficiency of the conjugation is independent of concentration between 2-8 µM of the protein (Figure S9). Furthermore, 25 equivalents of the probe can be utilized without interference of significant non-directed side-reaction (Figure S10). We attribute the ability of using higher concentrations of both protein and probe without significant observation of non-directed reaction to the relatively slow kinetics of the non-directed reductive amination reaction. Modification of therapeutic antibodies. Several antibodies have been approved as cancer therapeutics,16 including the IgG1 antibodies , rituximab (MabTheraTM), cetuximab (ErbituxTM), and the antibody mention above, trastuzumab (HerceptinTM) . 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 AzideProbe 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 the both the Azide- and Azo-Probes (Figures S11-S13). One of the major assets of these probes is that the NTA groups enable separation of the unmodified, mono-labeled and di-labeled 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 performs similarly in the affinity-guided 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

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 Azide-Probe 1 followed by SPAAC reaction with DBCO-Cy5 for visualization of the protein conjugates (Figure 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 non-guided conjugation that would lack the desired areaselectivity. Serotransferrin conjugates. We further investigated the impact of the labeling on the inherent metal-binding irontransporting protein serotransferrin. The protein was conjugated 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 10X 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 area-selective 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 metal-binding protein with a dedicated metalbinding pocket, we turned to the labeling of inherent metal-coordinating proteins that chelates the metal through surface exposed residues.



Page 4 of 11

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 Azide-Probe 1 and DBCO-Cy5 (10 eq.). The same gel was scanned for Cy5 fluorescence (left) and stained for protein by SimpleBlue SafeStain (right). (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 o-aminophenol-PEG10K using K3Fe(CN)6 as an oxidant. The analysis of the band intensities is shown in Figure S25.

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 were compared using anion exchange chromatography (Figure 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.

with DBCO-PEG20.000, yielding approximately 52-65% conversion, in accordance with 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 the number of HPLC purifications needed to isolate the modifiable conjugate.

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


Page 5 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 mono-labeled 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).

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 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 metal-binding 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 Fabdomain in the intact antibody.22

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 anilinemodified 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 Fabdomains. 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 Fab-domains (Tz: Figure 3b, lane 1. Rx, Cx: Figure S26). In order to investigate if the labeling is in fact

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 confirm 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.



Page 6 of 11

Figure 4. Performance of the conjugates and visualization of cell surface receptors. (a) SPR sensorgrams for unmodified, purified mono-labeled 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 uncertainty 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.

protein A to the Fab-domain,24 which inactivates the antigen-binding 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 (Figure 4a and S33-35), but importantly the conjugates retained sub-nanomolar affinity, thus, showing highly preserved binding affinity.

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 Amodified chip. The extracellular domain of the EGFR is used to assess the Kd-values, which shows similar affinities of the conjugates and unmodified cetuximab (Figure 4a and S30-32). This shows that the conjugation chemistry has no effect on the affinity of these conjugates for their target.

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

The trastuzumab antibodies are not compatible with the protein A-modified chips, presumably due to binding of


Page 7 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry 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 area-selectivity 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 DNA-templated 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.

thioredoxin-tag 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 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 Azide-Probe 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 10X excess of the unmodified antibody relative to the conjugate show a markedly 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.

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 20-1000 pmol. Generally, final concentrations 2-6 µM of proteins were used. The equivalents have been calculated according to the protein content. 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) was 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).

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 Azo-Probe 2, reductive cleavage and labeling with oaminophenol-RhoB, 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 similar to the Cy5modified conjugates prepared with the Azide-Probe 1.

*Tris-buffer (20 mM, pH 8.5) for HPLC or direct MALDI-TOF analysis. Cleavage buffer (Azobenzene: 50 mM phosphatebuffer, 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 x TM TM TM 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 flow rate of 0.5 mL/min with a gradient of 0-90% B over 17 min. Nanobody conjugates were purified on a Thermo ScienTM TM TM tific Dionex DNAPac PA-100 4x250 mm column using a Hewlett Packard Agilent 1100 Series HPLC system. The purification was performed with Buffer A (25 mM Tris) and Buffer B (25 mM Tris, 1 M NaCl) with an increasing gradient of buffer B (0-75%) over 10 min (flow rate: 1 mL/min).

CONCLUSION In summary, we have developed efficient probes for areaand chemoselective conjugation to native metal-binding and his-tagged 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



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 1030 min). The concentration was determined using absorbance ® at 280 nm measured on a ND-1000 NanoDrop spectrophotometer.

Page 8 of 11

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 iron-saturated (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 pre-treating the cells with 1 uM native holo-Tf 5 min before addition of holo-Tf-Cy5.

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 MilliQ-water. The protein solution (1 µL) was mixed with a 2% TFA in MilliQ-water solution (1 µL) and DHAP-matrix (1 µL).29 The mixture was mixed and spotted on the MALDI plate.

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.

Labeling of the azide-modified conjugates with DBCOreagents. To the azide-modified conjugates in PBS was added the DBCO-reagent (10 eq.). The reaction mixture was incubated at rt for 2 h to overnight. The excess reagent was removed using an Amicon Ultra® centrifugal filter (MWCO 1030 kDa, 14100 g for 40 min) or the reaction mixture was directly subjected to analysis by SDS-PAGE (NuPAGE 4-12% Bis-Tris).

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 ºC. For the competition experiment, cells were charged with 1 uM unmodified rituximab 5 min before incubation. Cells were washed three times in PBS by centrifugation at 300xg for 5 min. 200.000 cells were then analyzed by flow cytometry. To enable microscopy, 400.000 cells in 50 µl PBS were incubated in 8-well chamber slides for 30 min at 37 ºC. Prior to incubation, cell chambers were treated with 0.05 mg/ml poly-L-lysine (>30,000 Da, Sigma) for 10 min at 37 ºC and washed twice with PBS. Cells were subsequently fixed and stained according to the protocol used for transferrin uptake experiments.

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 manufactures instructions). Remaining Na2S2O4 was removed by repeated washes in an Amicon Ultra® centrifugal filter (MWCO 30 kDa, 14100 g for 10-30 min).

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 1h at 37 ºC. For the competition experiment, cells were charged with 1 µM unmodified cetuximab 5 min before incubation. Cells were subsequently washed three times in PBS and fixed and stained according to the standard protocol.

The coupling conditions between the aniline and o-amino14 phenol was described by Francis and co-workers. Briefly; To the protein (2-20 µM), in a phosphate-buffer (50 mM, pH 6.5), was added the o-aminophenol-compound (20-200 µM, 10 eq.) and K3Fe(CN)6 (200-2000 µM, 100 eq.). The reaction was performed over 25 min at rt. The reaction mixture was diluted with phosphate-buffer and excess reagents removed by repeated washes in an Amicon Ultra® centrifugal filter (MWCO 10-30 kDa, 14100 g for 40 min).

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 ºC. 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 PBS, vortexed and analysed by flow cytometry.

Study of site-selectivity using hinge region cleavage studies. 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 manufacturers 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 ug/mL), and kept at 37 ºC in humidified air containing 5% CO2.

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 20X/0.8 objective and a 63X/1.4 Oil DIC immersion objective (Zeiss). DAPI fluorescence was excited with the 405 nm line,

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


Page 9 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 linie. Digital image recording was performed with the ZEN 2012 software (Zeiss) and image analysis was performed using Fiji (ImageJ).

reference flow cell no antibody was captured. Then, 2-fold dilution series (10 nM - 0.625 nM) were injected for 180 sec (association) and the dissociation followed for 300 sec. The sensor surface was regenerated with a 30 sec injection of 10 mM glycine-HCl (pH 2.5) after each run.

Flow cytometry. Flow cytometric analysis was performed on a Gallios Flow Cytometer (Beckman Coulter) using the 638 nm laser. Maximum 10.000 cells were assessed in each experiment. Forward and side scatter was used to gate a homogenous population of single cells. FCS files were analyzed using Kaluza.

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 sec) and dissociation (1200 sec) was 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 sec.

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 hours. The tryptic peptides were micro-purified using Empore™ SPE Disks of C18 30 octadecyl packed in 10 µl pipette tips.

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.

Mass spectrometry. NanoESI-MS/MS analyses were performed on eksigent nanoLC 415 system (Sciex) connected to a TripleTOF 6600 mass spectrometer (Sciex) equipped with a NanoSpray III source (AB Sciex). The micro purified 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 ReproSil-Pur C18-AQ 3 μm resin (Dr. Marisch GmbH, Ammerbuch-Entringen, 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 Da respectively. To identify the site of modification, the Azo-mod (full or hydrolyzed) was set as variable modifications of Lysine residues. All sites of Azomodification were manually inspected and validated. Spectra with modified peptides were extracted by importing all data with an ion score cut-off of 30 and a significant threshold of 31 0.01 (p

Small-Molecule Probes for Affinity-Guided Introduction of

Recommend Documents