Simultaneous and independent dual site-specific self-labeling of

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Simultaneous and independent dual sitespecific self-labeling of recombinant antibodies Carolin Wollschlaeger, Ivo Meinhold-Heerlein, Xiaojing Cong, Karen Braeutigam, Stefano Di Fiore, Felix Zeppernick, Torsten Klockenbring, Elmar Stickeler, Stefan Barth, and Ahmad Fawzi Hussain Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00545 • Publication Date (Web): 05 Oct 2018 Downloaded from http://pubs.acs.org on October 6, 2018

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Bioconjugate Chemistry

Simultaneous and independent dual site-specific self-labeling of recombinant antibodies

Carolin Wollschlaeger1§, Ivo Meinhold-Heerlein2§, Xiaojing Cong3, Karen Bräutigam4, Stefano Di Fiore5, Felix Zeppernick2, Torsten Klockenbring1, Elmar Stickeler6, Stefan Barth7,8# and Ahmad Fawzi Hussain6#*

1Department

of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology

and Applied Ecology IME, Forckenbeckstrasse 6, 52074, Aachen, Germany. 2Department

of Gynecology and Obstetrics, University Hospital Giessen, Justus-Liebig-University

Giessen, Klinikstr. 33, 35392 Giessen, Germany 3Institute

of Chemistry, - Nice, UMR 7272 CNRS - University Côte d'Azur, 06108 Nice cedex 2,

France 4Department

of Gynecology and Obstetrics, University Hospital Schleswig-Holstein, Campus

Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. 5Fraunhofer

Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6,

52074, Aachen, Germany. 6Department

of Gynecology and Obstetrics, University Hospital RWTH Aachen, Pauwelsstrasse

30, 52074, Aachen, Germany. 7Medical

Biotechnology and Immunotherapy Unit, Institute of Infectious Disease and Molecular

Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa. 8South

African Research Chair in Cancer Biotechnology, Department of Integrative Biomedical

Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa.

* Correspondence: Ahmad Fawzi Hussain Dept. of Gynaecology and Obstetrics University Hospital RWTH Aachen, Pauwelsstr. 30, 52074, Aachen, Germany. Tel. +49-241-80 37925, Email: [email protected]

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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

Abstract Antibody-based diagnostic and therapeutic reagents armed with effector molecules such as dyes and drugs offer hope in the battle against cancer. Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a singlepot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye®700 and Alexa-Fluor®647. This dual-labeled antibody bound to EGFR+ ovarian cancer cell lines and tissue samples with high specificity and its phototherapeutic efficacy was confirmed by the selective killing of EGFR+ cells in vitro.

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Introduction Antibody-based diagnostic and therapeutic agents have revolutionized cancer management by improving the effectiveness of tumor detection and treatment. Antibodies recognize specific antigens and achieve therapeutic efficacy via several mechanisms, such as blocking specific pathways, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity 1. The genetic modification of antibodies can improve their innate properties and also add functionality, yielding new classes of antibody-based diagnostic and therapeutic agents by arming the antibodies with effector molecules such as fluorescent proteins and other fluorophores, toxins, nanoparticles, radionuclides and drugs. Antibody-drug conjugates (ADCs) and antibody-radionuclide conjugates (ARCs) are two classes of antibody conjugates that overcome the major limitations of traditional tumor detection and therapy. These agents combine the binding specificity of antibodies with the therapeutic activity of cytotoxic drugs or the detection potential of radionuclides. There are currently two ADCs on the market, namely brentuximab vedotin (trade name Adcetris) and trastuzumab emtansine (trade name Kadcyla), which are indicated for the treatment of certain CD30+ lymphomas and HER2+ breast cancer, respectively, as well as one ARC (111In capromab pendetide, trade name ProstaScint) which is used to monitor prostate cancer 2. The success of these products has encouraged academic and industry researchers to fill the development pipelines with additional antibody conjugates. Most antibody conjugates currently under development are produced by randomly conjugating the effector molecules to the antibody using either the reduced sulfhydryl groups of cysteine residues or the amino groups of lysine side chains. This strategy generates a heterogeneous population of conjugates with a range of drug to antibody ratios (DARs) 3. In contrast, site-specific conjugation targets a unique site for conjugation, generating homogeneous conjugates with the potential for higher efficacy, more predictable pharmacokinetic behavior and greater tolerability 4. Various site-specific conjugation methods have been developed to generate homogeneous antibody conjugates with defined pharmacokinetic, therapeutic and safety profiles, but they tend to be complex and expensive because multiple reactions are required 5-6. In contrast, self-labeling proteins such as the SNAP and CLIP tags provide antibodies with controlled conjugation sites that can be labeled in a single reaction. The SNAP and CLIP tags are derivatives of the human O6alkylguanine-DNA alkyltransferase (AGT), which has the ability to conjugate benzylguanine (BG) or benzylcytosine (BC) molecules, depending on the folding pattern 7. We previously used SNAP and CLIP tags to develop a simple, controlled and robust method for the generation of antibody

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conjugates by fusing the tag to single-chain variable fragment (scFv) antibodies 8-11. However, this method only allows one effector molecule to be attached to the antibody. Given the potential for acquired resistance to available ADCs, and the failure of many ADC candidates in clinical trials due to adverse effects and limited efficacy, there is a high unmet medical need for antibodies equipped with multiple drugs or with both drugs and imaging reagents, allowing the precise evaluation of biodistribution, therapeutic efficacy and adverse effects in preclinical studies. Here, we describe a site-specific dual conjugation method which simultaneously yet independently allows the conjugation of two different effector molecules to an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425). The single-pot reaction can be carried out under physiological conditions, achieving high labeling efficiency and a constant DAR. The phthalocyanine dye IRDye®700DX (IRDye700) and Alexa Fluor®647 were chosen as the effector molecules because they have distinct optical imaging properties, allowing the quantitative evaluation of conjugation efficacy, cell binding and internalization. The photodynamic activity of IRDye700 dye allowed us to determine the therapeutic efficacy of the ADC against four ovarian cancer cell lines, and the visualization of both effector molecules allowed us to detect cell lines in vitro as well as primary cancer cells in ex vivo tissue samples.

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Results: Simulation In all the three fusion protein simulations, the structures of both the CLIP tag and SNAP tag were well maintained compared to the crystal structure of alkyltransferase (Cα RMSD < 2 Å). The scFv showed slightly larger fluctuations, but mostly preserved the same fold as the template structure (Cα RMSD ranging from 2.9 ± 0.2 Å to 3.2 ± 0.4 Å). Notable secondary-structure changes occurred in the first two β-strands of the light chain, which were converted into β-bend structures in the simulations (Figure 1 and Supplementary Material 1). However, the Debye-Waller factor of this region was also high in the template crystal structure, indicating high flexibility.

Figure 1: Secondary structures of the fusion protein during the simulations initialized from three different models.

Dual site-specific labeling The dual labeling of the CLIP-scFv-425-SNAP fusion protein with BC-647 and BG-PEG24IRDye700 was evaluated by photometry, using the extinction coefficients of the dyes and the theoretical extinction coefficient of the proteins. We achieved a conjugation efficiency of 86% for the CLIP tag and 84% for the SNAP tag individually, while the conjugation efficiency was 89% for the CLIP tag and 85% for the SNAP tag in combination. Furthermore, the site-specific conjugation activity of the tags with the BC/BG-modified dyes was confirmed by irreversibly blocking each tag with specific blocking reagents. These reagents completely abolished the conjugation of BC-647

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and BG-PEG24-IRDye700 to both CLIP-scFv-425-SNAP and SNAP-scFv-425-CLIP fusion proteins (Figure 2). The independent dual site-specific labeling of the fusion protein was confirmed by conjugating BC647 and BG-PEG24-IRDye700 to the CLIP and SNAP tags of the CLIP-scFv-425-Cat-SNAP fusion protein simultaneously. Digestion of the fusion protein with cathepsin B was expected to yield a large CLIP-scFv-425 fragment with only Alexa 647 fluorescence and a small SNAP-tag fragment with only IRDye700 fluorescence. As shown in Figure 3B, SDS-PAGE analysis by in-gel fluorescence scanning indicated the presence of two bands of larger size with Alexa 647 fluorescence (red signal) and two more abundant and a low abundant digestion product of smaller size with IRDye700 fluorescence (green signal), as well as a band representing the undigested protein (yellow signal). The twin bands probably reflect the removal of the flexible (GGGGS)3 linker between the antibody light-chain and heavy-chain variable domains

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and the low specificity of

Cathepsin B enzyme, which is similar to the Cat-linker peptide sequence. These results confirmed that the SNAP and CLIP tags can specifically and independently self-conjugate with BG-IRDye700 and BC-647, respectively.

Figure 2: Antibody dual labeling using SNAP and CLIP tags. Schematic diagram of the bicistronic eukaryotic expression cassette for the CLIP-scFv-425-SNAP (A) and SNAP-scFv-425- CLIP (B) fusion proteins. (C) Detection of fluorescent signals representing BC-647 and BG-PEG24-IRDye700 dual-labeled protein following SDS-PAGE. The CLIP-scFv-425-SNAP and SNAP-scFv-425- CLIP fusion proteins were incubated respectively with a two-fold molar excess of BC-647 and BG-PEG24-IR700 (1 and 4); a five-fold molar excess of the SNAP-tag blocking reagent bromothenylpteridine (BTP), followed by a two-fold molar excess of BC-647 and BG-PEG24-IRDye700 (2 and 5); a five-fold molar excess of CLIP-tag blocking reagent

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followed by a two-fold molar excess of BC-647 and BG-PEG24-IRDye700 (3 and 6) After separation by SDS-PAGE the Alexa Fluor®647 and IRDye700 labelled proteins were visualized using the CRi Maestro multispectral imaging system. The yellow band shows dual labeling; the red band represents Alexa Fluor®647 and the green band represents BG-PEG24-IR700.

Figure 3: Site-specific dual labeling of the CLIP and SNAP tags. (A) Schematic diagram of the CLIP-scFv425-Cat-linker-SNAP fusion protein expression cassette. (B) The CLIP-scFv-425-Cat-linker-SNAP protein was conjugated to BC-Alexa Fluor®647 and BG-PEG24-IR700, then incubated with 0.25 µg human cathepsin B for 2 h at 37°C (1) or with the buffer as a control (2). The yellow band shows the dual labeling of CLIPscFv-425-Cat-linker-SNAP, the red bands represent digestion products of the CLIP-scFv-425 labeled with Alexa Fluor®647 and the green bands represent digestion products of the SNAP-tag labeled with BG-PEG24IR700. The fusion proteins were visualized after separation by SDS-PAGE using the CRi Maestro multispectral imaging system. (C) Protein Marker, Broad Range (7−175 kDa) stained with Coomassie Brilliant Blue.

Flow cytometry and confocal microscopy The dual-labeled CLIP-scFv-425-SNAP and SNAP-scFv-425-CLIP fusion proteins showed clear binding to the three EGFR+ cell lines (SKOV-3, OVCAR-3 and IGROV-1) and was thus able to distinguish between EGFR+ and EGFR– cells. A fluorescent signal was detected by flow cytometry in all EGFR+ cell lines (SKOV-3, OVCAR-3 and IGROV-1) after incubating them with the dual-

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labeled CLIP-scFv-425-SNAP, SNAP-scFv-425-CLIP or scFv-425-SNAP fusion proteins, but no signal was detected in the EGFR– cell line A2780 under the same experimental conditions (Figure 4A). The flow cytometry results revealed that both dual-labeled CLIP-scFv-425-SNAP, SNAPscFv-425-CLIP are retained the full binding activity of scFv-425 against EGFR and inserting SNAP-tag or CLIP-tag at N-terminal dose not significantly effect scFv-425 binding affinity. Furthermore inserting SNAP-tag or CLIP-tag at N-terminal dose not significantly effect scFv-425 binding affinity, therefore CLIP-scFv-425-SNAP fusion protein was used for performing the further experiments. The cellular uptake of the dual-labeled fusion protein (and scFv-425-SNAP-VG) was investigated by confocal microscopy. Both the Alexa 647 (red) and VG (green) fluorescence signals were detected intracellularly in EGFR+ ovarian cell lines treated with 647-CLIP-scFv-425-SNAP-VG (Figure 4B). Under the same experimental conditions, only VG fluorescence was observed in EGFR+ cells after incubating them with scFv-425-SNAP-VG (Figure 4C). Neither of the fusion proteins was detectable in the EGFR– cell line A2780 after incubation for 2 h at 37°C.

Figure 4: In vitro activity of the CLIP-scFv-425-SNAP and SNAP-scFv-425-CLIP dual-labeled proteins. The specific binding activity of labeled proteins to EGFR was evaluated by flow cytometry (A) and confocal microscopy (B and C). (A) The binding activities of 647-CLIP-scFv-425-SNAP-VG, VG-SNAP-scFv-425CLIP-647 and scFv-425-SNAP-VG were determined using four ovarian cancer cell lines by flow cytometry. Gray filled curves represent untreated cells; black curves represent cells treated with 1 µg/mL scFv-425SNAP-VG, dotted black curves represent cells treated with 1 µg/mL 647-CLIP-scFv-425-SNAP-VG; gray

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curves represent cells treated with 1 µg/mL VG-SNAP-scFv-425-CLIP-647. (B and C) Internalization analysis of the CLIP-scFv-425-SNAP and scFv-425-SNAP fusion proteins by fully automated confocal microscopy. Ovarian cancer cell lines were incubated with 1 µg/mL 647-CLIP-scFv-425-SNAP-VG (green, red and yellow signals) (B) or with 1 µg/mL scFv-425-SNAP-VG (green signal) (C) at 37°C for 2 h followed by 20 min incubation with Hoechst 33342 fluorescent nuclear counterstain(blue signal). Scale bar = 10 μm.

Binding of the EGFR-specific fusion protein to ovarian tumor samples The clinical potential of the CLIP-scFv-425-SNAP fusion protein was confirmed by investigating its ability to distinguish between ovarian tumor and healthy tissues. The CLIP-scFv-425-SNAP protein bound specifically to the ovarian cancer tissues but no binding was detected when normal ovarian tissues were treated with the same fusion protein. Similar results were observed when the ovarian cancer tissues were treated with scFv-425-SNAP. This confirms that adding the CLIP-tag to scFv-425-SNAP had no impact on the binding activity of the antibody (Figure 5).

Figure 5: Ex vivo immunohistochemistry binding studies of CLIP-scFv-425-SNAP and scFv-425-SNAP to ovarian cancer tissue sections and healthy controls. Ovarian cancer biopsies were incubated with 1 µg CLIP-scFv-425-SNAP (A) or scFv-425-SNAP (B), whereas normal ovarian biopsies were treated with 1 µg CLIP-scFv-425-SNAP (C). An anti-SNAP antibody was used as the primary antibody and an AP-labeled goat-anti-mouse monoclonal antibody was used as the secondary antibody.

Photoimmunotoxicity The phototoxic effects of 647-CLIP-scFv-425-SNAP-IRDye700 and scFv-425-SNAP-IRDye700 associated with the exposure of ovarian cancer cell lines to NIR light were evaluated using an XTT-based colorimetric cell proliferation assay. Dose-dependent cytotoxicity was observed in all EGFR+ ovarian cancer cell lines treated with 647-CLIP-scFv-425-SNAP-IRDye700 or scFv-425SNAP-IRDye700 followed by illumination with NIR light. The 50% elimination of the ovarian cancer cells (IC50) values for 647-CLIP-scFv-425-SNAP-IRDye700 were 45 nM (SKOV-3), 50 nM (OVCAR-3) and 60 nM (IGROV-1), whereas those for scFv-425-SNAP-IRDye700 were 52 nM

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(SKOV-3), 66 nM (OVCAR-3) and 70 nM (IGROV-1). A2780 cells remained viable even when treated with 400 nM of the fusion proteins. No significant toxicity was observed after treating the cells with the fusion proteins under the same conditions in the absence of NIR light treatment (Figure 6).

Figure 6: Cytotoxicity of 647-CLIP-scFv-425-SNAP-IRDye700 and scFv-425-SNAP- IRDye700 fusion proteins toward three EGFR+ and one EGFR– ovarian cancer cell lines. The phototoxicity of 647-CLIP-scFv425-SNAP-IRDye700 (▼), scFv-425-SNAP- IRDye700 (■) and 647-CLIP-scFv-425-SNAP-IRDye700 without light treatment (▲) were evaluated using an XTT cell proliferation assay. The results indicate percent of cell viability normalized against untreated control cells. Data are presented as means ± standard error of the mean (SEM) and obtained from three replicate experiments each using triplicate samples.

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Discussion Cancer detection and management have been significantly improved by the development of targeted therapies, especially those based on antibodies. Three antibody conjugates (two ADCs and one ARC) have already been approved for clinical use and several more are in the development pipeline

13.

These treatment options can improve patient safety, survival and life

quality, compared to conventional chemotherapy, and can also reduce overall treatment costs by avoiding off-target effects. Dual antibody conjugation approaches that equip antibodies with more than one type of effector molecule have recently been reported

14-17.

These approaches could overcome the limitations of

current antibody-based diagnostic and therapeutic agents, such as low therapeutic/diagnostic activity and acquired resistance. Furthermore, the conjugation of imaging agents to these probes will allow their biodistribution to be mapped accurately and any adverse effects to be investigated in preclinical studies. In a previous study, the EGFR1-specific antibody panitumumab and the Her2-specific antibody trastuzumab were conjugated to indocyanine green (ICG)-sulfo-OSu and [111In] using the chelator diethylene triamine penta-acetic acid (DTPA)

16.

The same principle has been applied to a dual-

labeled diagnostic probe for breast cancer: trastuzumab was conjugated to IRDye 800 and [111In]DTPA using a three-step coupling method

18.

Both methods relied on traditional protein

conjugation, which lacks the specificity of site-specific labeling and does not incorporate effector molecules with a defined DAR. Recently, the intraoperative imaging properties of three antibodies were investigated after dual labeling with a radionuclide and fluorophore at DARs of 0, 1, 1.5, 2 and 3

19.

DARs of 1–1.5

achieved optimal imaging in this study, whereas antibodies with higher or lower DARs accumulated to unacceptable levels in the liver or to insufficient levels in the tumor 19. Furthermore, the conjugation site on the antibody also affects the toxicity, pharmacokinetic properties and therapeutic efficacy of antibody conjugates 5, 14, 20. To avoid the issues discussed above, several dual site-specific conjugation methods have been developed to generate antibody conjugates with defined pharmacokinetic properties, therapeutic indices, and safety profiles. However, these methods require multiple reaction steps and the associated manufacturing costs are therefore high. Recent attempts to overcome these challenges include the engineering of cysteine and selenocysteine residues at specific sites based on THIOMAB and SELENOMAB technology. These methods allow the attachment of iodoacetamide and methylsulfone-modified effector molecules to cysteine and selenocysteine

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residues with near uniform stoichiometry 21-23. Another approach involves orthogonal conjugation methods based on transglutaminase and engineered cysteine, respectively. This method was used to equip trastuzumab with two different fluorophores, which were conjugated to the antibody by introducing a strained alkyne (BCN) and thiol, by deglycosylation, transglutaminase-mediated conjugation, reduction and re-oxidation steps

17.

Another appealing approach is controlled

disulfide rebridging using water-soluble allyl sulfones. This method overcomes the limitations of natural cysteine residues and the limited water solubility and reactivity of current disulfide rebridging reagents

24.

Although these strategies allow the efficient conjugation of effector

molecules to antibodies and generate nearly homogeneous products, they involve several reaction and purification steps and therefore the production costs are commensurately increased. Compared to the aforementioned approaches, our method does not need a reduction step, separate conjugation steps or the changing of the conjugation buffer due to the intrinsic monovalent conjugation properties of both SNAP-tag and CLIP-tag which allow a rapid one-step reaction, site-directed and autocatalytic labeling under physiological conditions with a uniform DAR 25-26. Furthermore, a broad range of BG/BC-modified fluorophores, thiol-reactive derivatives including BG/BC-Maleimide, amino reactive BG/BC-NHS or BG/BC-NH2 building blocks are commercially available and this facilitate modifying various effector molecules and constantly increasing the range of SNAP-tag and CLIP-tag applications. Our molecular dynamics (MD) simulations revealed that the single-chain antibody, SNAP tag and CLIP tag retained their native folding propensity and structure in the context of the fusion protein. These results also showed that the SNAP tag and CLIP tag self-conjugation sites do not interfere with the function of the antibody and serve as effective spacers between the antibody and the effector molecules, thus eliminating any chemical and physical impact of direct contact with the antibody polypeptide. Our flow cytometry, confocal microscopy, immunohistochemistry and toxicity assays yielded comparable results. There were no significant differences in cytotoxicity between scFv-425-SNAP and CLIP-scFv-425-SNAP. Importantly, the SNAP and CLIP tags were conjugated simultaneously and independently to two different labels in a one-pot reaction. The inclusion of a blocking reagent specific for one of the two tags completely abolished the corresponding conjugation reaction. Furthermore, digesting 647-CLIP-scFv-425-Cat-SNAP-IRDye700 with cathepsin B yielded two groups of protein fragments of different sizes, each carrying one of the two fluorescent dyes (Figure 4). These data confirm that simultaneous and site-specific labeling occurred on both tags. The incorporation of the CLIP tag reacted with the dye VG did not significantly affect the antibodyantigen binding reaction of the fusion protein or the intensity of the fluorescent signal. This was

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confirmed by comparing the binding activity of the dual-labeled protein with scFv-425-SNAP-VG by flow cytometry and confocal microscopy (Figure 4). Consistent with previous studies

27,

we found that our construct was not only able to selectively

detect ovarian cancer cell lines with different EGFR expression profiles but also to distinguish between the tissues of ovarian cancer patients and healthy ovaries. Additionally, IRdye700antibody conjugates can be activated using harmless NIR-light to produce cytotoxic reactive oxygen species that kill treated cells by inducing cell necrosis

28-29.

Here, our construct was able

to reduce the viability of EGFR+ ovarian cancer cells in a dose-dependent manner. It is important to note that the key difference between our dual site-specific method and earlier technologies is that our method is not restricted to the labeling of full-size antibodies and is therefore compatible with antibody fragments such as the scFv described herein, and all other recombinant antibody formats. Antibody fragment are advantageous because their small size allows them to penetrate solid tumors efficiently and they are also rapidly cleared from the body. The SNAP and CLIP tags increase the molecular weight of the antibody from ~30 kDa to 75 kDa, which provides a good balance between tumor penetration and renal clearance 30. The next step in the development of this technology is to test the dual-labeled antibody in vivo, which will help to determine its stability, biodistribution and tumor accumulation, as well as its safety profile, pharmacokinetic properties and targeting efficiency. This study describes an efficient and easy method for the dual labeling of an antibody based on the use of self-labeling protein tags. To our knowledge this work demonstrate for the first time the use of SNAP-tag and CLIP-tag to simultaneous and specific labeling of individual protein (scFv) with two different effector molecules. Taken together the outcome of our initial in vitro and ex vivo experiments provide a sound evidence that this site-specific conjugation method can generate homogeneous antibody-based therapeutics and/or imaging probes equipped with dual functional agents.

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Experimental Procedures Simulation Initial models of the fusion protein were generated by homology modeling using Modeller v9.9 31. The X-ray crystal structures of alkyltransferase (PDB ID: 1EH6) and the Fab fragment of matuzumab (PDB ID: 3C09) were used as templates. From 20,000 models, we selected the three with the lowest discrete optimized protein energy (DOPE) score

31,

which feature different initial

positions of the three protein components. The PROPKA webserver

32

was used to examine the

asparagine and glutamine side-chain rotamers and to predict the histidine protonation states. Each model was then immersed in a truncated octahedron simulation box of explicit aqueous solution neutralized with ~30 mM NaCl. The box size ensured a minimum 20-Å distance between adjacent images of the protein under periodic boundary conditions. Each system consisted of ~160,000 atoms, including ~50,000 water molecules, 47 Na+ and 30 Cl– ions. The molecular simulations were carried out using Gromacs v4.6 field

34,

the TIP3P water model

35

33.

The Amber99SB-ildn force

and the Joung-Cheatham parameters

36

were used for the

protein, the water, and the ions, respectively. The systems were energy-minimized and then gradually heated to 300 K in six steps of 100-ps MD simulations (from 2 K to 60 K, 120 K, 180K, 240 K and 300 K). The velocities were generated consistent with a Maxwell-Boltzmann distribution at the corresponding temperature. Each system then underwent 100-ns MD simulations in the NPT ensemble (P = 1 bar, T= 300 K) applying the Andersen-Parrinello-Rahman barostat 37-38 and the Nose-Hoover thermostat 39. The LINCS algorithm 40 was used to constrain all the bond lengths, allowing a 2-fs time step. Van der Waals and short-range electrostatic interactions were cut off at 12 Å. Long-range electrostatic interactions were computed using the Particle Mesh Ewald summation (PME) 41 method with a Fourier grid spacing of 1.2 Å. The first 10 ns of the simulation was discarded for equilibration. DSSP was used to analyze the secondary structure of the fusion protein throughout the trajectory 42. Cell culture The human ovarian adenocarcinoma cell lines OVCAR-3 (ATCC HTB-161), SKOV-3 (ATCC HTB77), IGROV-1 (CVCL 1304), A2780 (ECACC 93112519) and the human embryonic kidney cells HEK-293T (ATCC CRL-11268) were cultured in RPMI-1640 medium supplemented with 2 mM Lglutamine, 10% (v/v) fetal bovine serum (FBS), and 100 U/mL penicillin−streptomycin. The cells were incubated at 37°C under a 5% CO2 atmosphere and relative humidity > 90%. The medium and additives were purchased from Invitrogen, Darmstadt, Germany.

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Protein expression and purification The CLIP-tag DNA sequence was either inserted upstream of the scFv-425-SNAP-tag DNA sequence using the NheI and SfiI sites or downstream of SNAP- scFv-425 DNA sequence using the XbaI and BlpI sites in the eukaryotic expression vector pMS (based on pSecTag2, ThermoFisher Scientific, Waltham, MA). This generate CLIP-scFv-425-SNAP and SNAP-scFv425- CLIP expression cassettes (Figure 2 A). The cathepsin B-cleavable peptide linker GGGGSALAL (Cat-linker) 12, 43 was inserted at the NotI and XbaI sites immediately downstream of the scFv-425 sequence but upstream of the SNAP-tag sequence to generate the CLIP-scFv-425-Cat-linker-SNAP cassette (Figure 3 A). After transfecting HEK-293T cells with pMS-CLIP-scFv-425-SNAP, pMS-SNAP-scFv-425- CLIP and CLIP-scFv-425-Cat-linker-SNAP using the RotiFect transfection reagent (Carl Roth GmbH, Karlsruhe, Germany), the CLIP-scFv-425-SNAP, SNAP-scFv-425- CLIP, CLIP-scFv-425-Catlinker-SNAP and scFv-Ki4-SNAP 11 fusion proteins were purified from the supernatant using a NiNTA Superflow cartridge (Qiagen, Hilden, Germany) on an Äkta fast protein liquid chromatography (FPLC) system (GE Healthcare Europe GmbH, Freiburg, Germany) as previously described 9, 44. Antibody dual labeling The benzylguanine linker BG-PEG24-NH2 (Covalys Biosciences AG, Witterswil, Switzerland) was chemically conjugated to the IRDye®700DX NHS ester infrared dye (LI-COR Biosciences GmbH, Bad Homburg, Germany) using N-hydroxysuccinimide ester-amino group chemistry. The resulting BG-PEG24-IRDye700

product

was

analyzed

and

purified

by

high-performance

liquid

chromatography (HPLC) as previously described 8, 45. The CLIP-scFv-425-SNAP and SNAP-scFv-425- CLIP fusion proteins were dual labeled with BGPEG24-IRDye700 and benzylcytosine (BC)-modified Alexa Fluor®647 dye (BC-647; Covalys Biosciences AG) by incubating the fusion proteins with a two-fold molar excess of each fluorescent dye in phosphate buffered saline (PBS) for 2 h in the dark at room temperature. Labeled proteins were purified from residual dyes using Zeba Spin desalting columns, 7K MWCO (Thermo Fisher Scientific). To verify the site-specific conjugation activities of the CLIP and SNAP tags, the fusion proteins was incubated for 1 h in the dark at room temperature with a five-fold molar excess of blocking reagents specific for either the SNAP-tag or the CLIP-tag (Covalys Biosciences AG). The blocked proteins were then incubated with a two-fold molar excess of BC-647 and BG-PEG24-IRDye700 for 2 h in the dark at room temperature. The labeling specificity was analyzed by SDS-PAGE with in-gel fluorescence scanning using the CRi Maestro imaging system (CRi, Woburn, MA, USA).

15



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The self-labeling efficiency of the CLIP and SNAP tags individually and in combination was determined by Nanodrop One

(ThermoFisher Scientific). The protein theoretical extinction

coefficients were calculated as previously described

46.

The simultaneous but independent

labeling of both the SNAP tag and CLIP tag was confirmed by labeling CLIP-scFv-425-Cat-linkerSNAP with BC-647 and BG-PEG24-IRDye700. The labeled protein was then digested with cathepsin B (Enzo Life Sciences, Lörrach, Germany). Briefly, 10 µg of fusion protein labeled with BG-Vista Green dye (BG-VG; Covalys Biosciences) was incubated with 0.25 µg cathepsin B in digestion buffer (0.2 M NaCl, 1 mM EDTA, 10 mM dithiothreitol, 0.25 M sodium acetate, pH 5.5) 47

for 2 h at 37°C. After separation by SDS-PAGE, the fusion proteins were visualized using the

CRi Maestro imaging system. Flow cytometry The narrow near-infrared (NIR) fluorescence peak of IRDye700 by cannot be detected by traditional flow cytometry and confocal microscopy, so the BG-PEG24-IRDye700 was replaced with BG-VG for flow cytometry and confocal microscopy experiments. A FACSCalibur device was used with the corresponding software (Becton & Dickinson, Heidelberg, Germany) to evaluate the binding of dual-labeled CLIP-scFv-425-SNAP and SNAP-scFv-425- CLIP to EGFR+ ovarian cancer cell lines (OVCAR-3, SKOV-3, IGROV-1) and the EGFR– cell line (A2780). We incubated 1 µg/mL of either 647-CLIP-scFv-425-SNAP-VG, VG-SNAP-scFv-425- CLIP-647 or scFv-425SNAP-VG with 4×105 cells in 200 μL PBS for 30 min on ice. After washing the cells twice with 1.8 mL PBS in a conventional cell washer (UltraCW, Helmer Scientific, Noblesville, USA), the fluorescence intensity was measured by flow cytometry. Internalization assay Cells were visualized using an Opera QEHS confocal imaging system (PerkinElmer, Rodgau, Germany) with a 40x air objective. We seeded ~4 x 104 cells from each ovarian cell line in black 96-well flat-bottomed µclear plates (Greiner Bio-One, Frickenhausen, Germany), incubated them overnight under standard growth conditions. Following addition of 1 µg/mL of either 647-CLIPscFv-425-SNAP-VG or scFv-425-SNAP-VG the cells were incubated at 37°C for a further 2 h to promote internalization of the recombinant scFv ligands. The cell nucleus was stained with 5 µg/ml Hoechst 33342 a fluorescent nuclear counterstain (Thermo Fisher Scientific) for 20 min. The internalization of the labeled fusion proteins was measured by monitoring the Alexa-Fluor 647 and VG fluorescence signals. Immunohistochemistry using human tissue samples

16


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Human ovarian cancer biopsies were collected at the University Hospital RWTH-Aachen according to the guidelines of the ethical committee and written consent was obtained from the patients (EK 206/09). The formalin-fixed paraffin-embedded (FFPE) ovarian biopsies were prepared as previously described 48, blocked with 1% (v/v) bovine serum albumin in PBS for 1 h and incubated with either CLIP-scFv-425-SNAP or scFv-425-SNAP at 4°C overnight. The slides were washed with PBS and incubated with the M2D11 anti-SNAP antibody

49

for 3 h at room temperature. After washing the

slides with PBS, alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (GAMAP) (Dianova, Hamburg, Germany) diluted 1:50 in blocking solution was added and incubated overnight at 4°C. The slides were then washed twice with PBS and the AP activity was detected using naphthol ASBI phosphate (sodium salt, 0.5 mg/mL; Sigma-Aldrich, Taufkirchen, Germany) and New Fuchsin (0.1 mg/mL; Sigma-Aldrich) in 0.1 M Tris-HCl (pH 8.5). Levamisole (0.35 mg/mL; Sigma-Aldrich) was added to the reaction mixture to inhibit endogenous AP activity. Finally, the biopsies were counterstained using hematoxylin and eosin (Sigma-Aldrich) and images were captured using a Leica DMR-HC light microscope controlled by a Leica QWin software (Leica Microsystems, Wetzlar, Germany). Toxicity assays The toxicity of the 647-CLIP-scFv-425-SNAP-IRDye700 and scFv-425-SNAP-IRDye700 proteins against EGFR+ ovarian cancer cell lines (OVCAR-3, SKOV-3 and IGROV-1) and the EGFR– cell line (A2780) was determined using the XTT II cell proliferation kit (Roche, Mannheim, Germany) as previously described 8, 27, 50. All experiments were performed at least three times in triplicates, and the data are presented as means ± standard error of the mean (SEM). The reagent concentration required to eliminate 50% of the ovarian cancer cells (IC50) relative to control cells was determined using GraphPad Prism v5 (GraphPad, San Diego, CA).

17



Conflicts of interest: The authors declare that they have no conflict of interest.

Supporting Information Movie legends Movie 1: Structures of CLIP-scFv-425-SNAP fusion protein. Movie 1: Structures of scFv-425 protein.

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Figure 1: Secondary structures of the fusion protein during the simulations initialized from three different models. 338x190mm (300 x 300 DPI)



Figure 2: Antibody dual labeling using SNAP and CLIP tags. Schematic diagram of the bicistronic eukaryotic expression cassette for the CLIP-scFv-425-SNAP (A) and SNAP-scFv-425- CLIP (B) fusion proteins. (C) Detection of fluorescent signals representing BC-647 and BG-PEG24-IRDye700 dual-labeled protein following SDS-PAGE. The CLIP-scFv-425-SNAP and SNAP-scFv-425- CLIP fusion proteins were incubated respectively with a two-fold molar excess of BC-647 and BG-PEG24-IR700 (1 and 4); a five-fold molar excess of the SNAP-tag blocking reagent bromothenylpteridine (BTP), followed by a two-fold molar excess of BC-647 and BG-PEG24-IRDye700 (2 and 5); a five-fold molar excess of CLIP-tag blocking reagent followed by a two-fold molar excess of BC-647 and BG-PEG24-IRDye700 (3 and 6) After separation by SDS-PAGE the Alexa Fluor®647 and IRDye700 labelled proteins were visualized using the CRi Maestro multispectral imaging system. The yellow band shows dual labeling; the red band represents Alexa Fluor®647 and the green band represents BG-PEG24-IR700. 139x91mm (600 x 600 DPI)


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Figure 4: In vitro activity of the CLIP-scFv-425-SNAP and SNAP-scFv-425-CLIP dual-labeled proteins. The specific binding activity of labeled proteins to EGFR was evaluated by flow cytometry (A) and confocal microscopy (B and C). (A) The binding activities of 647-CLIP-scFv-425-SNAP-VG, VG-SNAP-scFv-425-CLIP647 and scFv-425-SNAP-VG were determined using four ovarian cancer cell lines by flow cytometry. Gray filled curves represent untreated cells; black curves represent cells treated with 1 µg/mL scFv-425-SNAPVG, dotted black curves represent cells treated with 1 µg/mL 647-CLIP-scFv-425-SNAP-VG; gray curves represent cells treated with 1 µg/mL VG-SNAP-scFv-425-CLIP-647. (B and C) Internalization analysis of the CLIP-scFv-425-SNAP and scFv-425-SNAP fusion proteins by fully automated confocal microscopy. Ovarian cancer cell lines were incubated with 1 µg/mL 647-CLIP-scFv-425-SNAP-VG (green, red and yellow signals) (B) or with 1 µg/mL scFv-425-SNAP-VG (green signal) (C) at 37°C for 2 h followed by 20 min incubation with Hoechst 33342 fluorescent nuclear counterstain(blue signal). Scale bar = 10 μm.



Figure 5: Ex vivo immunohistochemistry binding studies of CLIP-scFv-425-SNAP and scFv-425-SNAP to ovarian cancer tissue sections and healthy controls. Ovarian cancer biopsies were incubated with 1 µg CLIPscFv-425-SNAP (A) or scFv-425-SNAP (B), whereas normal ovarian biopsies were treated with 1 µg CLIPscFv-425-SNAP (C). An anti-SNAP antibody was used as the primary antibody and an AP-labeled goat-antimouse monoclonal antibody was used as the secondary antibody.


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Figure 6: Cytotoxicity of 647-CLIP-scFv-425-SNAP-IRDye700 and scFv-425-SNAP- IRDye700 fusion proteins toward three EGFR+ and one EGFR– ovarian cancer cell lines. The phototoxicity of 647-CLIP-scFv-425SNAP-IRDye700 (▼), scFv-425-SNAP- IRDye700 (■) and 647-CLIP-scFv-425-SNAP-IRDye700 without light treatment (▲) were evaluated using an XTT cell proliferation assay. The results indicate percent of cell viability normalized against untreated control cells. Date are presented as means ± standard error of the mean (SEM) and obtained from three replicate experiments each using triplicate samples. 190x155mm (300 x 300 DPI)



Table of Contents Graphic 169x136mm (300 x 300 DPI)


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Simultaneous and independent dual site-specific self-labeling of

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