O6-alkylguanine-DNA transferase (SNAP) as capture module for site-specific covalent bioconjugation of targeting protein on nanoparticles Serena Mazzucchelli,*a Miriam Colombo,b Elisabetta Galbiati,b Fabio Corsi,a Josè M. Montenegro,c

Wolfgang J. Parak,c Davide Prosperib

a Sacco Hospital, via G. B. Grassi, 74, Milan 20157, Italy; b University of Milan- Bicocca, piazza della Scienza, 2, Milan, 20126, Italy; c Philipps-Univ. Renthof 7, Marburg, 35037, Germany.

ABSTRACT

A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the biologically mediated site-specific covalent conjugation of targeting proteins to nanoparticles. ScFv800E6, an scFv mutant selective for HER2 antigen overexpressed in breast cancer cells was chosen as targeting ligand. The fusion protein SNAP-scFv was irreversibly immobilized on magnetofluorescent nanoparticles through the recognition between SNAP module and pegylated O6-alkylguanine derivative. The targeting efficiency of the resulting nanoparticle against HER2-positive breast cancer cells was assessed by flow cytometry and immunofluorescence.

Keywords: magnetic nanoparticles, fusion protein, capture module, targeting module, bioconjugation, scFv, tumor targeting, breast cancer

1. INTRODUCTION

The need for new technologies to produce bioactive organic-inorganic hybrid nanomaterials with control on physical and biochemical properties is now widespread in fields such as material science, biophysics, molecular biology, pharmacology and molecular medicine, as also testified by the large number of papers devoted to proposing novel methods, which appeared in top journals in the last few years1-3. When using nanoparticles (MFN) for targeted cancer diagnosis, one of the main issues concerns the design and optimization of effective functionalization strategies to achieve an efficient targeting of specific cell receptors4-6. One of the biggest problems in designing MFN functionalized with peptides and proteins to optimize molecular recognition, lies in the possibility to accurately control the orientation ligand on the surface of the nanoparticles7-8. So far, three main approaches have been followed to finely control ligand orientation: 1) site-specific immobilization on MNP is mediated by a small peptide or protein which exploits high affinity toward targeting protein8, 2) conjugation is mediated by an affinity tags commonly utilized for protein purification and inserted at genetic level in protein primary sequence 9,10 and 3) site-specific immobilization can occur via chemoselective ligation.11 In this paper, we present the potential of a new method based on the genetic encoding of SNAP fusion proteins capable of irreversibly cross-reacting with a suicide inhibitor (pegylated O6-alkylguanine derivative) anchored to the solid surface for the reliable biofunctionalization of nanoparticles. This approach has several advantages compared with other current methods: 1) the reaction is fast and irreversible, 2) a complete control on the site of conjugation of the protein (thus, on the protein orientation on the nanoparticle surface) can be achieved, 3) the reaction works best in a biocompatible environment and 4) the reaction is byproduct-free, so that each purification step is simple and efficient. The method was validated using a SNAP-scFv mutant of the anti-HER2 antibody against breast cancer cells, demonstrating that the resulting scFv-functionalized nanoparticles were able to selectively bind to the specific membrane receptors expressed on target-positive cancer cells. Our method is rapid, efficient and potentially applicable to most proteins, especially to the increasingly popular scFv targeting ligands, without apparent loss of biological activity, which usually represents a severe obstacle in the fabrication of bioinspired hybrid materials. Another feature of attractiveness of our method is that it makes use of hydrophobic nanoparticles, which have been rendered water-soluble by coating with a suitable amphiphilic polymer. In

Colloidal Nanocrystals for Biomedical Applications VIII, edited by Wolfgang J. Parak, Marek Osinski, Kenji Yamamoto, Proc. of SPIE Vol. 8595, 859502 · © 2013 SPIE

CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2001648

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this way, the same approach can be immediately extended to several kinds of nanomaterials, including gold, quantum dots, and many others that are prepared by solvothermal decomposition methods in organic solvents. The work we are now presenting is strongly interdisciplinary, offering a new strategy for the efficient, site-specific and orientation-controlled immobilization of proteins on the surface of nanoparticles, useful for both chemists and biologists.

2. METHODOLOGY

2.1 Synthesis of PMA-coated Fe3O4 fluorescent nanoparticles (MFN1) 1.0 M fluoresceinamine (0.5 mL in DMSO) was added to a 0.5 M PMA solution in CHCl3 (5 mL) and incubated overnight at room temperature.12 The resulting fluorescent PMA (63 μL) was added to MNC13 (4.6 mg in CHCl3), homogenized and the solvent was then evaporated at reduced pressure. Sodium borate buffer (SBB, pH 12, 10 mL) was added obtaining nanoparticle dispersion (MFN1). MFN1 were concentrated in Amicon tubes (cutoff 100 kDa) by centrifuging at 3500 rpm (1 h), washed two times with SBB and concentrated (each centrifuge cycle was 20 min at 3500 rpm) to a final volume of 200 μL.

2.2 Synthesis of L1-functionalized magnetofluorescent nanoparticles (MFN2) MFN1 (2.25 mg in 1 mL) was incubated with 0.1 M EDC (1.8 μL) for 2 min. Then 5.7 μL NH2-ended O6-PEG-guanine (2 mM, in deionized water) was added, shaken for 2 h, washed two times with water and finally concentrated by centrifuging at 3500 rpm for 10 min.

2.3 Conjugation of SNAP-scFv to MFN2 Purified SNAP-scFv (0.3 mg) with MFN2 (2 mg) in PBS, pH 7.4 (1 mL), in the presence of 1 mM DTT, were incubated overnight at 4 °C. Next, unconjugated SNAP-scFv was removed by centrifuging the mixture in amicon YM-100 tubes and the concentrated particles were washed three times with PBS (5 mL).

2.4 Construction of the expression vector SNAP tag DNA was amplified from pGEX-6P-1-SNAP-GFP vector (kindly provided by Petra Hohenberger, Karlsruher Institut für Technologie (KIT)), using these primers: 5'-CCGAGAATTCATGGACAAAGACTGCGAAATG-3' (forward primer) and 5'-CGTAGAATTCGCCCAGCCCAGGCTTGCCCAG-3' (reverse primer), containing an EcoRI site (underlined). The PCR product was digested with EcoRI and ligated to the corresponding site of the expression vector pPICZαA-scFv800E6.14 The ligation mixture was electroporated into E. coli DH5α competent cells for propagation of the recombinant plasmid. The resulting recombinant expression vector pPICZαA-SNAP-scFv was confirmed by restriction endonuclease digestion and DNA sequencing.

2.5 Strains and plasmids E. coli DH5α was used as a host strain for propagation of the plasmids vector. P. pastoris KM71H (arg4; aox1::ARG4) and GS115 (his4) (Invitrogen) were used as hosts for expressing SNAP-scFv gene. Plasmid pPICZαA-scFv800E6 was used for constructing the plasmid vector.14

2.6 Transformation in P. pastoris and screening of transformants The recombinant expression vector pPICZαA-SNAP-scFv was linearized with HindIII and the digested product was transformed into the P. pastoris host strains KM71H and GS115 by electroporation (1.5 kV, 400 Ω, 25 μF; Bio-Rad Gene Pulser). Transformants were selected on YPDS (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol) plates containing 100 μg mL–1 Zeocin. For small-scale screening of SNAP-scFv-productive clones, single clones from YPD agar plates were grown in 5 mL YPD medium at 30 °C overnight with shaking at 250 rpm. The cultures were centrifuged at 1500×g for 4 min and then the pellets resuspended in 5 mL of BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% YNB, 0.00004% biotin, 0.004% histidine, 0.5% methanol) or BMDY (BMMY containing 2% dextrose instead of methanol). Methanol was added to the culture medium every 24 h at a final concentration of 0.5%, in order to maintain induction. After 48 h, the cultures were centrifuged at 5000×g for 10 min and the supernatants were analyzed by SDS-PAGE and Western blotting as described below. For medium-scale screening of SNAP-scFv fusion protein-productive clones, single clones from YPD agar plates were grown in 10 mL YPD medium at 30 °C overnight with shaking at 250 rpm. The cultures were centrifuged at 1500×g for 4 min and then the pellets

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resuspended in 10 mL of BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% YNB, 0.00004% biotin, 0.004% histidine, 0.5% methanol) or BMDY (BMMY containing 2% dextrose instead of methanol). Induction was maintained adding methanol to the culture medium every 24 h at a final concentration of 0.5%. After 0, 24, 48 and 72 h induction, the cultures were centrifuged at 5000×g for 10 min and the supernatants analyzed by SDS-PAGE and Western blotting.

2.7 Optimization of induction conditions In order to setup the optimal conditions for SNAP-scFv expression, medium-scale culture experiments were performed in different induction conditions. The best producing clone GS115-pPICZαA-SNAP-scFv-1 was grown in 10 mL YPD medium at 30 °C overnight with shaking at 250 rpm. The cultures were centrifuged at 1500×g for 4 min and then the pellets were resuspended in 10 mL of BMMY with different methanol concentration (0.5% and 2%) and in BMMY supplemented with 2% dextrose or 0.8% glycerol. The cultures, yielding an initial OD600 value of 10, were supplemented every 24 h with methanol to a final concentration of 0.5% or 2% to maintain induction. Culture supernatants were sampled at different times to monitor SNAP-scFv production by Western blot analysis as described below.

2.8 Purification of SNAP-scFv 200 mL yeast culture in BMMY with 2% dextrose yielding an initial OD600 value of 10 was induced by daily addition of methanol to a final concentration of 0.5%. After 48 h, the culture supernatant was filtered through 0.22 µm filters, dialyzed overnight in 50 mM sodium phosphate pH 8.0, 300 mM NaCl and loaded onto a Ni-NTA Agarose (Qiagen) column (bed volume 0.5 mL) pre-equilibrated with 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10 mM imidazole. The column was washed twice with 10 volumes of 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 20 mM imidazole and the protein eluted with a stepwise imidazole gradient, 100 mM to 200 mM, in the same buffer. Fractions were collected and analyzed by SDS-PAGE and Western blotting as described below. Protein content was determined using the Coomassie Plus Protein Assay Reagent from Pierce and bovine plasma immunoglobulin G as the standard protein.

2.9 Analysis by SDS-PAGE and Western blot SDS-PAGE was performed according to Laemmli using 12% (v/v) polyacrylamide gels.15 The proteins were detected by Coomassie Brilliant Blue R-250 staining or transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore) for Western blotting analysis with horseradish peroxidase (HRP)-conjugated anti-myc antibody (Invitrogen). Target proteins were visualized using an enhanced chemiluminescence detection system (GE Healthcare).

2.10 Dot blot assay Dot blot was performed by filtering proteins and/or nanoparticles onto PVDF membranes, utilizing a Manifold I dot blot apparatus (GE Healthcare), and incubating in blocking solution (5% skim milk in PBS, Tween 0.05%) for 1 h at RT. The membrane was then probed for 1 h at RT in blocking solution using rabbit anti-Myc-HRP antibody (Invitrogen) at a 1:5000 dilution. Membranes were rinsed thrice in 0.05% Tween in PBS for 10 min. Immunoreactive spots were revealed using ECL Western blotting reagent (GE Healthcare).

2.11 Cell Cultures MCF7 and MDA-MB-468 cell lines were used as HER2 positive and HER2 negative cellular models, respectively. Cells were cultured in 50% Dulbecco’s Modified Eagle’s Medium (DMEM) and 50% F12, supplemented with 10% fetal bovine serum, L-glutamine (2 mM), penicillin (50 UI mL–1) and streptomycin (50 mg mL–1) at 37 °C and 5% CO2 in a humidified atmosphere and subcultured prior to confluence using trypsin/EDTA. Cells culture medium and chemicals were purchased from EuroClone.

2.12 Flow cytometry Cells were cultured on a multiwell dish until subconfluence. Then, cells were incubated 1 h at 37 °C in the presence of SSMFN (20 μg mL–1 or 100 μg mL–1). Cells were treated for FACS analysis with standard methods. Labeled cells were analyzed on a FACS Calibur flow cytometer (Becton Dickinson). 20000 events were acquired for each analysis, after gating on viable cells.

2.13 Confocal Laser Scanning Microscopy Cells were cultured on collagene (Sigma) pre-coated coverglass slides until subconfluence. Cells were incubated 1 h at 37 °C with 100 μg mL–1 of SSMFN or with 20 μg mL–1 of free SNAP-scFv. Then, cells were washed twice with PBS,

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5' AOX

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fixed for 10 min with 4% paraformaldehyde (Sigma) and treated for 10 min with 0.1 M glycine (Sigma) in PBS. A blocking step was performed for 1 h at RT with a solution containing 2% bovine serum albumin (Sigma), 2% goat serum in PBS. SNAP-scFv was revealed by a FITC-conjugated antibody to whole murine IgG (MP Biomedicals) at a 1:300 dilution by incubating for 2 h at RT. Microscopy analysis was performed with a Leica SP2 AOBs microscope confocal system. Images were acquired with 63× magnification oil immersion lenses at 1024×1024 pixel resolution.

2.14 Cell death analysis MCF7 cells were cultured on a multiwell dish until subconfluence, then were incubated 24 h at 37 °C in the presence of SSMFN (20 μg mL–1, 50 μg mL–1 or 100 μg mL–1). After incubation cells were washed twice with PBS and treated for FACS analysis according to PE Annexin V Apoptosis Detection Kit I manufacturer’s protocol (Becton Dickinson Biosciences). Then cells were analyzed within 1 h on a FACS Calibur flow cytometer (Becton Dickinson). 20000 events were acquired for each analysis, after gating on viable cells. Therefore, we considered cell death as the populations positive for Annexin V and for 7AAD staining alone and together. The results are expressed as means ± standard deviation of the mean of 3 individual experiments.

2.15 Cell proliferation assay MCF7 cells were cultured on a 96 multiwell dish at a density of 5000 cells cm–1. Then cells were incubated with SSMFN (20 μg mL–1 and 100 μg mL–1). At the indicated time points, cells were washed with PBS and then incubated for 3 h at 37 °C with 0.1 mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) stock solution previously diluted 1:10 in DMEM medium without phenol red. At the end of the incubation, 0.1 mL of MTT Solubilizing Solution was added to each well to solubilize the MTT formazan crystals (Sigma-Aldrich). Absorbances were read immediately in a BIORAD Microplate reader using a test wavelength of 570 nm and a reference wavelength of 690 nm. The results are expressed as means ± standard deviation of the mean of 5 individual experiments.

3. RESULTS

ScFv is a small antibody fragment consists of variable regions VH and VL of a monoclonal antibody linked via a synthetic loop,16 and is usually obtained through genetic engineering with phage display technology to optimize the selectivity target.17 ScFv has improved biodistribution compared to intact IgGs due to small size (typically 20-30 kDa) and absence of an immunogenic Fc portion. However, their potential in cancer diagnosis and therapy is limited by a reduced affinity and specificity due to their monovalent binding. Recently we have published the production and purification of a scFv800E6 variant of anti-HER2 monoclonal antibody in Pichia pastoris.14 Here, in a pPICZαA/scFv800E6 vector we have insert SNAP DNA sequence at the N-terminal position using a EcoRI restriction site. The resulting pPICZαA/SNAP/scFv vector (schematically represented in Figure 1) was used to electroporate KM71H and GS115 P. pastoris host strains.

Figure 1. Schematic diagram of the pPICZαA-SNAP-scFv800E6 expression vector. The recombinant gene encoding SNAP tag was inserted into the pPICZαA-scFv800E6 vector under the control of the alcohol-oxidase-1 (AOX1) promoter, in frame with the prepro-α-factor signal sequence (αF) and with Myc and histidine (6×His) tags.

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MWM Wash Fr.1 Fr.2 Fr.3

117 kDa -

90 kDa -

49 kDa -

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25 kDa -

Not induced Induced

MWM 72h 48h 24h Oh 72h 48h 24h Oh

100 kDa KM71H clone 1

100 kDa KM71 H clone 5

100 kDa GS115 clone 11

100 kDa GS115 clone 1

Four SNAP-scFv expressing clones were selected for medium-scale culture experiment. Figure 2 reports that GS115/pPICZαA/SNAP/scFv-1 clone and KM71H/pPICZαA/SNAP/scFv-5 clone show a drop in protein expression after 72 h of induction at 30 °C, probably due to SNAP-scFv degradation in culture media, while GS115/pPICZαA/SNAP/scFv-11 clone and KM71H/pPICZαA/SNAP/scFv-1 show a undetectable and a very low SNAP-scFv expression, respectively. Therefore GS115/pPICZαA/SNAP/scFv-1 clone was chosen as best producer and used for optimization experiments. 48 h of induction with 0.5% methanol and 2% dextrose let us to obtain the highest expression level. Secreted SNAP-scFv with a C-terminal 6×His tag was purified from medium by a single purification step onto Ni-NTA agarose column in 1.5 mg L–1 yield (Figure 3).

Figure 2. Medium scale screening of P. pastoris producing clones. Equivalent amounts (10 µL) of culture supernatants from KM71H and GS115 SNAP-scFv productive clones after 0, 24, 48 and 72 h of induction were run in SDS-PAGE and analyzed by Western blotting with the anti-myc-HRP antibody. As a control, clones were grown under non-inducing conditions (without methanol).

Figure 3. Proteins from wash (Wash) and fractions (Fr.1, Fr.2 and Fr.3) obtained by Ni-NTA affinity chromatography were separated by SDS-PAGE and visualized by Commassie staining. MWM: molecular weight markers. Optimization of bulky protein accommodation and reduction of possible nonspecific adsorption of other biomolecules to MFN is achivied linking a PEG spacer (5 kDa) to a guanine functionality. The ligand L1, containing an NH2-ended O6-PEG-guanine, was synthesized in 3 steps according to the procedure previously described.18 Highly uniform 8 nm

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magnetite nanocrystals (MNC) coated with oleate surfactants were obtained by solvothermal decomposition in organic solvents as previously described.13 Chloroform suspended MNC were transferred to water phase by mixing with a 0.5 M solution of an amphiphilic polymer (PMA) in sodium borate buffer (SBB, pH 12). PMA was obtained by condensation of poly(isobutylene-alt-maleic anhydride), previously reacted with fluoresceinamine, and dodecylamine.28 Fluorescent and superparamagnetic PMA-coated nanoparticles (MFN1) were highly dispersible in aqueous environment. The residual carboxylic groups of the polymer coating was used to bound L1 via EDC activation (MFN2, Scheme 1). The resulting MFN2 were stable in PBS buffer resulting in a dark clean solution. MFN2 characterization was performed by DLS measurements of a mean hydrodynamic size of 145 ± 2 nm in PBS (pH 7.4, 5 μg mL–1).

Scheme 1. Synthesis of SSMFN. Nanoparticle conjugation of the fusion protein was achieved by reacting purified SNAP-scFv with MFN2 in 1 mM dithiothreitol (DTT) dissolved in PBS, pH 7.4. After overnight incubation at 4 °C, the mixture was centrifuged in amicon YM-100 tubes in order to remove unconjugated SNAP-scFv. The concentrated particles were washed three times with PBS, resulting in SNAP-scFv-functionalized MFN (SSMFN). Dot-blot assay using an anti-myc-HRP antibody on SSMFN confirm the presence of scFv (Figure 4). Moreover, DLS showed an increment in the hydrodynamic size (178 ± 7 nm) upon conjugation.

Figure 4. Dot-blot assay of SSMFN conjugated with SNAP-scFv. 5 μg of SSMFN and MFN (pegylated MFN1) were filtered on a PVDF membrane and probed with an anti-Myc-HRP antibody. Immunoreactive spots were revealed with an ECL substrate. 100 ng of SNAP-scFv were loaded as positive control (C+). Targeting efficacy of SSMFN was evaluated using HER2-positive MCF7 cells as cellular model. Two different concentrations of SSMFN (20 μg mL–1 and 100 μg mL–1, respectively) were incubated 1 h at 37 °C with MCF7 cells and

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I"ICKL- C its HER2+ cells HERZ- cells HER2+ cells

1 ISNAP -scFv SNAP -scFv SSMFN SSMFN -

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MCF7 SSMFN 0.02 mg mL-1

MCF7SSMFN0.1 mg mL-1

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with HER2-negative MDA-MB-468 cells (100 μg mL–1, negative control). Flow cytometry assay performed on MCF7 cells treated with SSMFN show an increase in percentage of cells in positive region (Figure 5), while MDA cells remained unlabeled after SSMFN treatment at 100 μg mL–1. Flow cytometry results suggests a SSMFN selective recruitment to HER2 expressing MCF7 cells. Moreover, data show that SSMFN-HER2 recognition was dose-dependent. Specificity of binding between SSMFN and HER2 receptor was confirmed by confocal laser scanning microscopy. 100 μg mL–1 of SSMFN were incubated in parallel 1 h at 37 °C with MCF7 and MDA-MB-468 cells. As a positive control, MCF7 cells were incubated 1 h with SNAP-scFv 20 μg mL–1 and revealed with a fitc-labeled secondary antibody to whole murine IgG. SSMFN and SNAP-scFv were localized at cell membrane of HER2-positive cells only, thus confirming that SSMFN cell interaction is mediated by a specific interaction with HER2 transmembrane receptor (Figure 6).

Figure 5. MCF7 cells were incubated 1 h at 37 °C with 20 μg mL–1 (red) and 100 μg mL–1 (green) of SSMFN. As a negative controls, MDA-MB-468 cells treated with 100 μg mL–1 of SSMFN (violet) and untreated MCF7 cells (blue) were reported.

Figure 6. HER2+ cells (MCF) and HER2– cells (MDA-MB-468) were incubated 1 h at 37 °C with SSMFN (100 µg mL–1). SNAP-scFv incubation with MCF7 and MDA cells was used as positive and negative controls, respectively. SNAP-scFv was revealed by a FITC-conjugated secondary antibody against whole murine IgG (green). Membranes were stained with DiD oil (red). Scale bar = 10 μm. Finally, SSMFN toxicity profile was evaluated by cellular death experiments and by proliferation assay. Cell death was assayed on MCF7 cells after incubation with 20, 50 and 100 μg mL–1 of SSMFN. Our results show that SSMFN were non toxic at these concentrations, which is significant for in vitro and in vivo use (Figure 7A). MTT proliferation profile of SSMFN treated cells at 20 μg mL–1 and 100 μg mL–1 is similar to untreated cells suggesting SSMFN safety in cell cultures (Figure 7B).

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Figure 7. A) Cell death assay with SSMFN. MCF7 cells were treated with SSMFN (20 μg mL–1, 50 μg mL–1 and 100 μg mL–1) for 24 h. Cell death was assessed by measuring the exposure of Annexin V and the incorporation of 7-aminoactinomycin D evaluated by flow cytometry. The percentage of cell death in untreated population was subtracted. The results are expressed as means ± S.D. of the mean of 3 individual experiments. B) Cell proliferation assay with SSMFN. MCF7 cells were treated with SSMFN (20 μg mL–1 and 100 μg mL–1) for up to 48 h. Cell proliferation was tested by measuring the conversion of MTT into formazan. CTRL represents untreated control. The results are expressed as means ± S.D. of the mean of 5 individual experiments.

4. CONCLUSION

In summary, this paper presents a new modular approach for the selective immobilization on nanoparticles of proteins for targeting specific cancer cells. This method is based on the production of the targeting protein, i. e. an scFv antibody variant, in fusion with SNAP. SNAP mediates a quick, effective and a site-directed reaction with a O6-PEG-guanine derivative anchored onto the nanoparticle surface, resulting in the formation of a new covalent bond between the SNAP-scFv and the nanoconjugate with release of a guanine molecule. This strategy for nanoparticle bioengineering had an high potential and was demonstrated, as a proof of principle, using an anti-HER2 scFv immobilized on multifunctional nanoparticles, which proved very selective in targeting HER2 antigen in breast cancer cells. Moreover this approach can be considered of general value for the development of targeted nanoparticles for biomedical applications, since PMA polymer can be exploited for transporting various kinds of nanoparticles to water phase.29

5. REFERENCE

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