SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants

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SNAP-tag technology: a useful tool to determine affinity constants and other functional parameters of novel antibody fragments Judith Niesen, Markus Sack, Melanie Seidel, Rolf Fendel, Stefan Barth, Rainer Fischer, and Christoph Stein Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00315 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 11, 2016

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Bioconjugate Chemistry

Title SNAP-tag technology: a useful tool to determine affinity constants and other functional parameters of novel antibody fragments

Judith Niesen †*, Markus Sack‡, Melanie Seidel †, Rolf Fendel †, Stefan Barth†§, Rainer Fischer †,‡ and Christoph Stein †

Affiliations: †

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany

‡

Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany

§

Permanent address of Stefan Barth:

University of Cape Town South African Research Chair in Cancer Biotechnology Institute of Infectious Disease and Molecular Medicine (IDM) Faculty of Health Sciences Department of Integrative Biomedical Sciences Anzio Road Observatory, 7925 Cape Town, South Africa

ACS Paragon Plus Environment


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Abstract Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O6-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives. TOC-graphic:


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Introduction Antibodies and antibody fragments as part of diagnostics or immunotherapeutics have several important functional parameters, including their specificity, serum half-life, immunogenicity, foldingstability, and especially the binding affinity.1, 2 The latter determines the overall binding strength of an antibody to its antigen and therefore strongly influences its retention time at the site of interest, such as a tumor cell. Antibody derivatives, such as the fragment antigen binding (Fab) and the single chain fragment variable (scFv), the latter solely comprising the variable domains of the immunoglobulin (IgG) heavy and light chains connected by a short polypeptide linker, have a lower avidity than the parent antibody but retain its binding specificity.3,4 The most widespread method to determine the affinity of antibodies is surface plasmon resonance (SPR) spectroscopy, typically using a Protein A chip to capture the antibody followed by the application of the corresponding antigen in the fluidic phase.5,

6

Fab kinetic constants have been assessed using methods based on Flexchip technology,

where the Fab is immobilized on a gold surface and the corresponding antigen is injected over the surface at a single concentration. Likewise, biotinylated antigens are captured on a streptavidin-coated surface and the Fab is injected at different concentrations.7 Other possibilities include enzyme-linked immunosorbent assay (ELISA) or flow cytometry with the soluble antigen or intact living cells. The latter can determine the equilibrium binding constant of antibodies directed against cellular receptors in their natural environment and under native conditions.8, 9 Antibody derivatives lacking the fragment crystallizable (Fc) domain are difficult to monitor by SPR spectroscopy. Therefore, the direct immobilization of analytes on the sensor chip surface by NHS (N-hydroxysuccinimide)/

EDC

(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)

hydrochloride 10

chemistry is often followed by applying the antibody derivative in the fluidic phase. However, this can limit the accessibility of the epitope due to the unpredictable orientation of analytes on the surface, and the folding of complex proteins can be inhibited by chemical coupling.11 In contrast, the SNAPtag technology facilitates an enzymatically site-specific covalent labeling or immobilization of antibody fragments such as scFvs in a 1:1 stoichiometry without inhibiting the function of the fusion partner.12 The SNAP-tag is a modified version of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase, which naturally removes alkyl residues from damaged DNA. As a fusion partner, the SNAP-tag achieves irreversible coupling to substrates containing O6-benzylguanine (BG).13 The SNAP-tag has been used for the fluorescent labeling of fusion proteins and the biofunctionalization of surfaces, e.g. for the immobilization of proteins on monolayers of streptavidin printed on glass surfaces, where the three-tag system SNAP-FLAG-His10 (SFH) was pre-biotinylated with BG-biotin. This system is compatible with any protein containing SFH.14 The SNAP-tag has also been used for nanoparticle biofunctionalization, e.g. coupling scFv-SNAP proteins to fluorescence-labeled silica nanobeads or magnetic multifunctional nanoparticles.15,

16

Numerous applications using BG-

fluorophore labeled scFv-SNAP fusion proteins are established, e.g. as in vivo imaging probes as well as part of photo-immunotherapy agents.17-21 Here we used scFv fragments scFv2112 and scFv1711, ACS Paragon Plus Environment


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derived from the Food and Drug Administration (FDA)-approved mAbs cetuximab and panitumumab, respectively, each of which binds specifically to the epidermal growth factor receptor (EGFR), a wellcharacterized target overexpressed on many solid tumors.22 Both svFvs have been genetically fused to the SNAP-tag and used to develop immunotoxins and human cytolytic fusion proteins with promising cytotoxic and apoptotic effects against a variety of EGFR+ solid tumor cells.

22, 23

EGFR plays an

essential role in cell proliferation, the inhibition of apoptosis, and the promotion of metastasis,

24, 25

and is therefore a promising target for diagnostic and therapeutic strategies in cancer research.17, 26, 27 As stated above, antibody fragments such as scFvs have a lower avidity than full-length antibodies due to their single binding domain.4, 28 The affinity constant can also decline when the scFv is part of a fusion protein.29 Here we present a promising novel tool that can determine the affinity of antibody fragments by combining SNAP-tag technology with SPR spectroscopy, circumventing the drawbacks mentioned above. The method was evaluated using a commercial kit allowing the reversible capture of biotinylated ligands and fusion proteins and in our case a BG-biotin coupling reaction was used. Both scFvs showed a high affinity in the nanomolar range after binding to the analyte, the EGFR extracellular domain (EGFRex). Furthermore, we used the SNAP-tag to evaluate the properties of novel antibody fragments in a variety of experimental tests including ELISA, flow cytometry and confocal microscopy, thus demonstrating their behavior in competition assays and direct or indirect specific binding assays using cell surface or immobilized antigen. The detection of soluble EGFR could help to determine the tumor burden or to detect tumor cells in the blood of cancer patients.


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Results and discussion Expression, purification and labeling of the scFv-SNAP fusion proteins The EGFR-specific scFv2112-SNAP and scFv1711-SNAP proteins were expressed in HEK 293T cells. Both constructs were purified by immobilized metal affinity chromatography (IMAC), yielding 5–10 mg of purified protein per liter of supernatant with a final purity of ~80–90%. The yield and purity were similar to other scFv-SNAP fusion proteins, such as scFvKi4-SNAP (~10 mg/L) and scFv(H22)-SNAP (~8 mg/L).15 SNAP-tag fusion proteins have also been expressed in prokaryotes such as Escherichia coli and yeasts such as Saccharomyces cerevisiae with lower yields.12 For example, the HER2-specific construct SNAP-scFv800E6 was expressed in the yeast Pichia pastoris with a yield of only 1.5 mg/L in the most stable strain, the others suffering extensive SNAP-scFv degradation in the culture medium.16 The two novel scFv-SNAP fusion proteins were also recovered with high purity after only one purification step and without further enrichment for high performing clones, which is important and advantageous for potential in vivo applications. With the SNAP-tag technology it is easily possible to test whether novel antibody fragments are functional and to ensure that they are appropriate candidates for therapeutic applications, e.g. as part of immunotoxins or cytolytic fusion proteins.18 Both scFv-SNAP fusion proteins were therefore coupled to BG-modified fluorescent dyes or to BG-biotin. As an example, purified BG-488 labeled scFv-SNAP fusion proteins are shown after separation by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in Figure 1. The SDS-PAGE demonstrates the purity of the protein and the activity of the SNAP-tag after successful coupling to BG-488 (lanes 2 and 3 show scFv1711-SNAP BG-488 and scFv2112-SNAP BG-488, respectively, after staining with Coomassie Brilliant Blue, and lanes 4 and 5 show the same constructs visualized by Cri Maestro imaging). The coupling reaction achieved a labeling efficiency of ~80% and a 1.5-fold molar excess of BG-dye or BG-biotin to protein was sufficient.



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Figure 1:

Figure 1: SDS-PAGE analysis of BG-fluorophore-labeled scFv1711-SNAP and scFv2112-SNAP. Lane 1: color pre-stained protein standard, broad range (11-254 kDa). Lanes 2 and 3: scFv1711-SNAP BG-488 and scFv2112-SNAP BG-488 after staining with Coomassie Brilliant Blue. The same scFv fusion proteins shown in lane 2 and 3 were also visualized by their BG-488 fluorescence using the Cri Maestro™ imaging system with the blue filter set and are shown in lane 4 (scFv1711-SNAP BG-488) and lane 5 (scFv2112-SNAP BG-488). Dye spectra were unmixed using Cri Maestro™ software 2.2.

In vitro binding analysis The specific binding activity of scFv2112-SNAP and scFv1711-SNAP was demonstrated by flow cytometry using both direct and indirect labeling. BG-488-coupled scFv-SNAP fusion proteins allowed the direct measurement of binding without further detection antibodies (Figure 2 A). For indirect analysis, the same scFv-SNAP fusion proteins were detected with a murine anti-SNAP antibody and a secondary phycoerythrin (PE)-conjugated goat anti-mouse IgG (Figure 2 B). Binding was analyzed using three different EGFR+ cell lines, two with high-level EGFR expression (A431 with 148,723 and MDA-MB-468 with 85,891 EGFR expression level) and one with a moderate expression level (L3.6pl with 21,290 EGFR expression on the surface) as previously reported.22 Both assays confirmed the binding specificity of both constructs with no evidence of nonspecific binding to the EGFR– cell line U937 (Figure 2). Higher mean fluorescence intensities (MFIs) were achieved by indirect analysis, probably due to the more intense fluorescence emission of the PE-labeled detection antibody compared to the BG-488-labeled fusion protein. Both constructs were applied in equimolar concentrations and showed nearly the same binding strength to the EGFR+ cell lines in both approaches. The higher MFIs could also reflect the availability of more binding sites at the Fc-part of the mAbs using a monoclonal primary and a polyclonal secondary antibody as indirect detection method.


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The binding activity of the novel scFv-SNAP fusion proteins was also analyzed by ELISA using the extracellular domain of the EGFR (EGFRex). This confirmed the suitability of the binding conditions for the BG-biotin-labeled scFv-SNAP fusion proteins compared to their binding activity on cell lines expressing EGFR on the surface. The microtiter plate was coated with the EGFRex antigen at a 1:2 dilution starting at 0.3 µg/µL. We observed the specific and antigen-concentration-dependent binding of scFv2112-SNAP-BG-biotin and scFv1711-SNAP-BG-biotin by detection with horseradish peroxidase-conjugated streptavidin (Figure 2 C). The ability of the fusion proteins to bind A431 cells was demonstrated in a live cell confocal microscopy assay using scFv2112-SNAP-BG-488 as a representative example. Following the incubation of the cells with scFv2112-SNAP-BG-488 for 30 min on ice, the A431 cell membranes were clearly stained (Figure 2 D). In addition to strong and specific binding, the internalization of both BG-488-labeled fusion proteins has already been demonstrated in a variety of EGFR+ cell lines by live cell imaging.22 The feasibility of coupling scFv-SNAP constructs to a variety of BG-modified fluorescent dyes can be exploited to study living cells directly e.g. by fluorescence-based confocal microscopy.30 Live cell imaging by confocal microscopy was carried out using chamber slides, so the cells could be measured directly on the microscope after incubation with the scFv-SNAP probe without transfer prior to visualization or staining. Both recombinant fusion proteins retained their binding specificity after fusion to the SNAP-tag without structural changes that might affect binding activity. Hence, the activity of the scFv was unaffected by fusion to the SNAP-tag or by coupling to the BG-fluorophore or BG-biotin, as demonstrated by flow cytometry, confocal microscopy and ELISA using recombinant EGFRex.



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Figure 2:

Figure 2: The direct and indirect measurement of scFv2112-SNAP and scFv1711-SNAP binding specificity was confirmed using different approaches. (A) First, 150 nM of scFv2112-SNAP (black curve) and scFv1711-SNAP (dashed curve) labeled with BG-488 showed specific binding to the three EGFR+ cell lines, A431, MDA-MB-468 and L3.6pl. Nonspecific binding was not observed on the EGFR– cell line U937. (B) Indirect detection of scFv2112-SNAP and scFv1711-SNAP specific binding to the same cell lines using an anti-SNAP antibody and a goat anti-mouse IgG PE conjugate. (C) Binding of scFv2112-SNAP (black curve) and scFv1711-SNAP (gray curve) coupled to BG-biotin using EGFRex-coated ELISA plates. The binding of scFv-SNAP BG-biotin fusion proteins to EGFRex was revealed following incubation with the peroxidase conjugated streptavidin substrate by reading the OD at 450 nm. The standard error of the means (±SD) is represented by error bars from at least three independent experiments each measured in triplicate. (D) Representative experiment showing the binding of scFv2112-SNAP BG-488 to A431 cells by confocal live cell imaging microcopy after incubation on ice for 30 min with 150 nM scFv2112-SNAP (scale bar = 10 µm).


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Competition analysis The parental antibodies of scFv2112 and scFv1711 (i.e. cetuximab and panitumumab) bind adjacent and presumably overlapping epitopes but not completely identical ones.31-33 The conformational epitope of cetuximab is known to partially overlap the ligand-binding region on EGFR domain III, whereas the panitumumab epitope has not been completely defined.32 The scFv-SNAP fusion proteins were tested against each other and their parental mAbs in competition assays to investigate potential changes in their binding properties in the scFv format. For flow cytometry measurements, we selected MDA-MB-468 cells because these were previously used for specific binding analysis. The cells were prepared and handled as described above for the binding studies. After an incubation step with a 10fold molar excess of the competitor, the cells were incubated with the appropriate BG-488-coupled scFv-SNAP probes directly or the biotin-labeled parental mAbs, which were detected using fluorescein isothiocyanate (FITC) – conjugated streptavidin. The competitive behavior of both scFvSNAP fusion proteins and parental mAbs showed the expected characteristics. The binding of scFv2112-SNAP and scFv1711-SNAP was essentially blocked if the cells were pre-incubated with the corresponding mAb (Figure 3 A). The binding of one scFv-SNAP fusion was also inhibited when the cells were pre-incubated with the other, but the inhibition was up to 20% weaker than that achieved by the homologous mAb (Figure 3 B and table 1). The parental mAbs inhibited each other’s binding as expected (Figure 3 C). Neither the mock-scFv-SNAP nor the mock-mAb competitors affected the binding of the scFv-SNAP fusion proteins or the parental mAbs (Figure 3). For cetuximab and panitumumab, comparable data were obtained by SPR spectroscopy and isothermal titration calorimetry (ITC), i.e. both mAbs inhibited the other’s binding to an extracellular soluble form of the EGFR.31



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Figure 3

Figure 3: Competitive inhibition of the binding of scFv2112-SNAP, scFv1711-SNAP and the parental mAbs measured by flow cytometry. EGFR+ MDA-MB-468 cells were incubated with a 10-fold molar excess of unlabeled cetuximab or panitumumab (A) and scFv2112-SNAP or scFv1711-SNAP (B), before washing and incubating the cells with a fixed concentration of BG-488-labeled scFv-SNAP (A and B). (C) The parental mAbs were also tested against each other to measure their competitive binding inhibition. The assay was also conducted using non-specific mock-scFv-SNAP (A and B) and mock-mAb (C) to evaluate the specificity of the competition.


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Table 1: Competitive flow cytometry assay – [%] inhibition (± SD) Construct

Competitor

Percent inhibition (± SD)

scFv2112-SNAP BG-488

scFv1711-SNAP

85.76 (± 9.93)

cetuximab

91.81 (± 9.75)

panitumumab mock-scFv-SNAP scFv1711-SNAP BG-488

95.94 (± 0.25) ɑ

scFv2112-SNAP

75.69 (± 15.09)

cetuximab

94.63 (± 0.04)

panitumumab mock-scFv-SNAP cetuximab-biotin

panitumumab mock-mAb

panitumumab-biotin

ɑ

cetuximab mock-mAb

ɑ

1.88 (± 2.54)

95.31 (± 0.07) ɑ

7.42 (± 5.15) 90.45 (± 2.41) 26.93 (± 4.32) 83.48 (± 12.59)

ɑ

4.65 (± 1.01)

mock-scFv-SNAP (anti-CD64-scFv-SNAP construct 15), mock-mAb (human anti-AMA-1 antibody,

anti-apical-membrane-antigen-, ID F17B; unpublished results) Likewise, we demonstrated the competitive behavior of the scFv-SNAP fusion proteins by ELISA using EGFRex as the antigen (Figure 4). In this indirect competitive immunoassay, scFv2112-SNAP (Figure 4 A) and scFv1711-SNAP (Figure 4 B) competed with each other for binding to EGRFex, and also showed competitive inhibition following pre-incubation with the corresponding parental mAbs. As described above, pre-incubation with mock-scFv-SNAP or mock-mAb did not affect the binding of scFv2112-SNAP or scFv1711-SNAP, confirming their specificity for EGFR. These data thus supported the prediction that cetuximab and panitumumab and their scFv derivatives have overlapping epitopes and bind to spatially collocated surface regions of the receptor.32,

34

Our results thus

confirmed the competitive behavior of the two new scFv-SNAP fusion proteins by flow cytometry and direct binding to EGFR+ cell lines, as well as indirect ELISA and binding to the extracellular EGFR domain.



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Figure 4:

Figure 4: Competitive ELISA using scFv2112-SNAP and scFv1711-SNAP labeled with BG-biotin. ELISA plates were coated with 100 ng/well EGFRex overnight at 4°C. After blocking with 2% (w/v) casein for 30 min, the competitors were incubated with the antigen before 0.25 µg scFv2112-SNAP BG-biotin (A) or scFv1711-SNAP BG-biotin (B) was added to the wells. Bound scFv-SNAP BGbiotin fusion proteins were detected with horseradish peroxidase-conjugated streptavidin. Mean absorbance values (±SD) at 450 nm are presented from at least three independent measurements in triplicates


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Affinity measurements The avidity of a mAb decreases when it is converted to an antibody fragment such as a scFv, and it is known that the affinity of scFvs can also decline in the context of fusion proteins because of conformational changes in the scFv caused by the fusion partner.4, 29 In this work the affinity of each of the novel scFvs was therefore compared to that of the other and the corresponding mAbs. Panitumumab has a higher affinity than cetuximab for EGFR although the published values determined by SPR spectroscopy vary, e.g. 50 pM for panitumumab and 200 or 390 pM for cetuximab.35-38 The affinity of panitumumab was determined using the soluble extracellular domain of EGFR (including the N-terminal signal sequence) derived from baculovirus-infected insect cells.39 The origin of the EGFR used to determine the affinity of cetuximab was not specified.40-42 Our SPR spectroscopy data revealed KD values of 1.25 nM for panitumumab and 1.67 nM for cetuximab (Table 2 and Figure 5). We solely used the extracellular domain of EGFR derived from A431 cells, which was expressed as a recombinant antigen in HEK 293T cells and purified by IMAC using the Cterminal His6-tag. To determine the binding affinity, the antibody was first captured on the chip surface containing immobilized Protein A followed by the injection of EGFRex and the detection of binding. Affinity constants have previously been determined after capturing each antibody onto the Protein A surface, and SPR spectroscopy was applied by binding panitumumab and cetuximab to an extracellular soluble form of EGFR also derived from A431 cells, and purified by immuno-affinity chromatography.31,

43, 44

The resulting KD values of 1.24 nM for cetuximab and 0.95 nM for

panitumumab were broadly consistent with our data. Cetuximab was also shown to bind with a KD value of 2.7 nM to a commercial form of soluble EGFR derived from NS-0 mouse myeloma cells.45 The range of published affinity constants may reflect the diverse expression systems used to produce the antigen and the variation in the experimental approach. Post-translational modifications such as glycosylation could have been an impact on the expressed protein/antigen, and there are substantial differences between the glycan profiles generated in insect and mammalian cells, with the latter most closely related to human glycans.46,47 Furthermore, the dimerization of EGFR may hinder access to the binding site or may induce conformational changes that inhibit intermolecular interactions between the mAb and epitope.48



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Figure 5:

Figure 5: Sensograms for cetuximab (A) and panitumumab (B) measured with a Protein A Chip. Five different concentrations of EGFRex are demonstrated (30, 15, 7.5, 3.75 and 1.88 nM) with different colors and the corresponding fits are shown in black. The response units (RU) are plotted against time (s). The data were processed using Biacore T200 evaluation software.

It is currently not possible to measure the affinity of scFv-SNAP fusion protein by reversible SPR spectroscopy due the inability to immobilize the antibody fragment on the chip surface. Therefore, the fusion proteins scFv2112-SNAP and scFv1711-SNAP were labeled with BG-biotin and the affinity was determined using the Biotin CAPture Kit (Figure 6 A). Unlike direct immobilization or biotin/streptavidin capture, this kit allows regeneration and the exchange of ligands between experiments (in our case scFv-SNAP fusion proteins) without changing the sensor chip. The Biotin CAPture Kit is based on a novel capture concept which allows the reversible capture of biotinylated molecules and generic regeneration because of the nature of the oligonucleotide interaction. The sensor chip CAP surface is based on a carboxymethylated dextran matrix with immobilized oligonucleotides. To create a specific sensor surface, the Biotin CAPture reagent is added, consisting ACS Paragon Plus Environment

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of streptavidin conjugated with the complementary oligonucleotide.49 In the subsequent step, the biotinylated ligand (SNAP-tag fusion protein) is injected, allowing the antibody-antigen interaction to be monitored. The signal represents the mass increase after the antigen binds to the immobilized scFv. Its intensity is thereby directly proportional to the bound mass on the chip surface and is reported in response units (RU) that are plotted against time (Figure 6). The equilibrium binding constant (KD) was calculated as 3.95 nM for scFv2112-SNAP (Figure 6 B) and 3.99 nM for scFv1711-SNAP (Figure 6 C). The association rate (ka) of each scFv-SNAP fusion protein was lower than that of the corresponding mAb (Figure 6 and Table 2), resulting in higher KD values for the parental mAbs. However, this outcome was anticipated due to the greater avidity of full-length antibodies compared to corresponding monovalent fragments. In our case, the affinity of the scFvs was only slightly lower than that of the parental mAbs. Similarly, the affinity of eight different EGFR-specific scFvs was recently compared to the corresponding Fabs and mAbs using a kinetic exclusion assay with A431 cells.50 The KD values of the scFvs ranged from 264 to 1 nM and the values for the corresponding mAbs ranged from 1.17 nM to 0.6 pM, e.g. the KD values for mAb and scFv C10 were 1.17 and 264 nM, respectively, whereas those for mAb and scFv P2/2 were 0.77 pM and 17.01 nM, respectively.50

Table 2: Affinity constants (KD values) of scFv2112-SNAP, scFv1711-SNAP and the parental mAbs determined by SPR spectroscopy Construct

ka (1/Ms)

kd (1/s)

[105]

[10-5]

scFv2112-SNAP

13.89

5.49

scFv1711-SNAP

18.74

cetuximab panitumumab

KD (nM)

Chi2 (RU2)

Rmax (RU)

Rmax (RU)

fit

theoretical

3.95

33.62

36.90

0.18

7.48

3.99

71.15

75.30

1.65

87.72

14.68

1.67

29.60

41.20

1.25

43.55

5.45

1.25

22.95

30.10

1.01



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Figure 6:

Figure 6: Four-step procedure to measure the binding affinity of scFv-SNAP fusions using the Biotin CAPture Kit (GE Healthcare) by SPR spectroscopy. After each measurement, the sensor chip can be regenerated (step IV). (A) The Biotin CAPture reagent contains streptavidin conjugated to a single-stranded DNA oligonucleotide complementary to the single-stranded DNA on the Sensor Chip CAP surface. (B) Sensograms for scFv2112-SNAP. (C) Sensograms for and scFv1711-SNAP. The evaluation was processed based on the Biacore T200 evaluation software and the response units are plotted against the time in seconds. ACS Paragon Plus Environment

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We also measured the KD values by flow cytometry directly on EGFR+ cells lines, as also demonstrated for other scFvs.8, 29,51, 52 We recorded KD values of 12.1 nM for scFv2112-SNAP (Figure 7 A) and 12.7 nM for scFv1711-SNAP (Figure 7 B), which were approximately three fold higher than the corresponding SPR data (Table 2). SPR and flow cytometry methods for affinity measurement have been compared for five different scFvs derived from a non-human yeast surface display library. The nanomolar KD values derived from SPR spectroscopy and flow cytometry experiments were in some cases nearly the same and in other cases either higher or lower, but only in the case of one scFv clone the difference in the values was greater than five-fold.53 Similarly, for cetuximab and panitumumab affinity constants measured by flow cytometry and SPR spectroscopy were compared, the former yielded in KD values of 5 nM for cetuximab and 4 nM for panitumumab in binding to A431 cells, which were also higher values than those determined by SPR spectroscopy.54 In summary, we demonstrated the affinity constant of the scFv-SNAP fusion proteins after binding to the recombinantly generated and purified extracellular domain of the EGFR and to cells expressing the receptor on their surface. The affinity constants were comparable for each scFv and varied from ~4 to ~12 nM depending on the assay. We also successfully evaluated a protocol to determine the affinity of scFv-SNAP fusions by SPR spectroscopy using the Biotin CAPture Kit and determined the optimal experimental conditions.



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Figure 7:

Figure 7: Measurement of equilibrium binding constants (KD) for scFv2112-SNAP (A) and scFv1711SNAP (B) by flow cytometry using concentrations series of each scFv. The highest MFI-value of the scFv-SNAP fusion proteins bound to EGFR+ cells (L3.6pl) was set to 100% and all other data points were normalized to this value. The experiments were carried out six times and KD values were calculated by fitting a nonlinear regression curve.


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Conclusions We evaluated the suitability of the SNAP-tag as a fusion partner for two novel EGFR-specific scFvs by measuring a variety of functional parameters. Both scFv2112-SNAP (derived from cetuximab) and scFv1711-SNAP (derived from panitumumab) were expressed at higher yields than other scFv-SNAP fusion proteins and both achieved specific and strong binding to EGFR+ solid tumor cell lines and soluble EGFR in vitro as determined by direct and indirect flow cytometry assays and indirect ELISA. Comparable binding behavior was observed by live cell imaging using fluorophore-coupled scFvSNAP fusions by fluorescence confocal microscopy. Competition between the scFvs and between each scFv and its parental mAb was demonstrated using EGFR+ cell lines and soluble EGFR by flow cytometry and ELISA. As expected, scFv2112-SNAP and scFv1711-SNAP inhibited each other’s binding, as previously shown for the corresponding parental mAbs, indicating they bind overlapping epitopes on the extracellular EGFR domain. Affinity is an important parameter for novel antibody fragments and it varies when the fragment is part of a fusion protein. We therefore measured the affinity constants with two different setups: a flow cytometry assay using EGFR+ solid tumor cells, and SPR spectroscopy using the soluble EGFR extracellular domain. To our knowledge, we have shown for the first time a SPR-based protocol based on the Biotin CAPture Kit that facilitates the regenerative measurement of scFv-SNAP fusion protein affinity constants. Accordingly, the affinity constants of the scFv-SNAP fusion proteins (~4 nM) were only slightly higher than those of the parental mAbs (cetuximab ~1.7 nM, panitumumab ~1.3 nM) and were all in the low nanomolar range, whereas KD values of ~12 nM were recorded by flow cytometry. Our study highlights a promising strategy to test the functionality of novel antibody fragments fused to the human SNAP-tag, as well as a convenient SPR-based assay to measure affinity, a key factor affecting the performance of antibody-based diagnostics and therapeutics. Although we used EGFR-specific antibody fragments as a case study, the SNAP-tag can be recombinantly fused to any antibody fragment and the protocol we have developed is therefore universally applicable.



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Experimental Procedures Cell culture and cell lines The pancreatic carcinoma cell line L3.6pl was derived from COLO357 cells.55 Other cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, USA) or the German Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig, Germany). A431 is an epidermoid carcinoma line (DSMZ-No. ACC91), U937 is a histiocytic lymphoma line (DSMZ-No. ACC-5), MDA-MB-468 is a triple-negative breast cancer line (ATCC-No. HTB-132) and HEK 293T is a human embryonic kidney cell line (ATCC-No. CRL-11268). All cell lines were cultured at 37°C in a humidified 5% CO2, 95% air incubator using RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, USA) supplemented with 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum and 100 U/mL penicillin-streptomycin.

Protein expression and purification The vector system, the construction and expression of scFv2112-SNAP and scFv1711-SNAP is described in detail elsewhere.22 Briefly, the sequence of each scFv was inserted at the SfiI/NotI site of the pMS expression vector system derived from pSecTag2 (Invitrogen, Thermo Fisher Scientific) for eukaryotic expression in HEK 293T cells. The vector contains a C-terminal SNAP-tag (New England BioLabs, Schwalbach, Germany).15,

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HEK 293T cells were transfected with up to 1 µg of vector

DNA using 3 µL RotiFect (Carl Roth GmbH, Karlsruhe, Germany) according to the manufacturer’s instructions. The transfected cells were cultivated as described above, with 100 µg/mL Zeocin™ in the medium for selection (Invitrogen, Thermo Fisher Scientific). Successfully transfected cells were identified by monitoring fluorescence emitted by the enhanced green fluorescent protein (eGFP) reporter.15, 22 His6-tagged scFv-SNAP fusion proteins were purified from the cell-free culture supernatants by IMAC, targeting the His6 tag.22 Aliquots of the scFv-SNAP fusion proteins were supplemented with dithiothreitol (DTT) to a final concentration of 1 mM and stored at –80°C, as previously described.22 The mRNA for the extracellular domain of EGFR was isolated from A431 cells using the RNA isolation NucleoSpin® RNA Kit II (Macherey-Nagel, Düren, Germany). First-strand cDNA was synthesized by reverse transcription using an oligo-dT primer (SuperScript™ III CellsDirect cDNA synthesis system, Invitrogen, Thermo Fisher Scientific) and was amplified with the EGFR genespecific primers EGFR-ex- for-NheI (5'-TGC GCTAGC ATG CGA CCC TCC GGG ACG GCC-3') and EGFR-ex-back-Not (5'-TGC GCGGCCGC GGA CGG GAT CTT AGG CCC ATT CG-3'). The resulting EGFR product was inserted into the expression vector for HEK 293T cells and purified by IMAC targeting the His6-tag as previously described.22


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Protein labeling The purified scFv-SNAP tag fusion proteins were labeled with BG-modified fluorophores and biotin as previously described.15 The scFv2112-SNAP and scFv1711-SNAP proteins were conjugated with SNAP-Surface® Alexa Fluor® 488 (BG-488) and SNAP-Biotin® (BG-biotin) (New England BioLabs) using a 1.5-fold molar excess of dye to protein. The labeling reaction was incubated for 3 h at room temperature and then overnight at 4°C. Residual unbound dye or biotin was removed using Zeba™ Spin 0.5-mL desalting columns (Thermo Fisher Scientific) or PD-10 columns (GE Healthcare, Freiburg, Germany) depending on the volume of the conjugated protein. The scFv-SNAP fusion proteins labeled with BG-fluorophores were visualized after separation by SDS-PAGE using the Cri maestro imaging system (Cri, Woburn, USA) and the BG-biotin-labeled scFv-SNAP fusion proteins were tested for biotin activity by flow cytometry. The BG-biotin-labeled proteins were detected on EGFR+ human cancer cell lines with a streptavidin–FITC conjugate (Thermo Fisher Scientific). The coupling efficacy was estimated using the theoretical extinction coefficient of the protein (scFvSNAP) and the extinction coefficient of the fluorescent dye (BG-488). The parental mAbs cetuximab and panitumumab were conjugated to biotin using the EZ-Link® Hydrazide Biotin kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Both mAbs were obtained for research-only use from the University Hospital Aachen, Germany.22

Flow cytometry and confocal microscopy Flow cytometry experiments were carried out using the BD FACSVERSE instrument and the corresponding software FACSuite vs1.05 (BD Biosciences, Franklin Lakes, USA). For the direct measurement of specific binding, 4 x 105 cells were incubated with 150 nM of scFv2112-SNAP or scFv1711-SNAP labeled with BG-488 in 100 µL PBS for 30 min on ice, then washed in a conventional cell washer before analysis. For the indirect measurement of specific binding, the same number of cells was incubated with unlabeled scFv2112-SNAP or scFv1711-SNAP for 30 min on ice, followed by incubation steps using an anti-SNAP antibody (M2D11) 57 and a goat anti-mouse IgG PE conjugate (Dianova, Hamburg, Germany). Competition flow cytometry analysis was carried out by pre-incubating the EGFR+ target cells with a 10-fold molar excess of unlabeled competitor (scFv-SNAP or parental mAb). A fixed amount of BG-488-labeled scFv2112-SNAP or scFv1711-SNAP, or biotin-conjugated parental mAb was added after 30 min when equilibrium binding should have been achieved. For detection, FITC-conjugated streptavidin (Jackson ImmunoResearch, West Baltimore, USA) diluted 1:500 in PBS, was incubated with the cells for 1 h at room temperature. The percentage inhibition was calculated as follows: % inhibition = 100 – (MFI at 10-fold molar excess inhibitor / MFI without inhibitor) x 100, according to Gray et al. 58



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Affinity constants (KD values) for both scFv-SNAP fusion proteins were determined by calibrated flow cytometry, as previously described.8,

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L.36pl cells were used for analysis and a series of

different concentrations from 15 to 0.001 µg/mL were prepared for each scFv-SNAP protein. The antiSNAP antibody M2D11 and PE-conjugated anti-mouse IgG antibodies were used for detection as above. The highest MFI was set to 100% in the evaluation and the remaining MFIs were normalized to this value. The experiments were carried out at least six times. The KD values were calculated by fitting a nonlinear regression curve in GraphPad Prism v5 (GraphPad Software, La Jolla, USA). To demonstrate binding by “live cell imaging”, A431cells were visualized with a Leica TCS SP8 confocal microscope (Leica Mi crosystems GmbH, Wetzlar, Germany). We seeded 2 x 105 cells/mL in eight-well chamber slides as previously described.22 Specific binding was measured by incubating the cells with the BG-488-labeled scFv-SNAP fusion protein for 30 min on ice.

ELISA High-binding ELISA plates (Greiner BioOne, Frickenhausen, Germany) were coated overnight at 4°C with different concentrations of the antigen (0.3 µg EGFRex, diluted 1:2) per well in 100 µL PBS. To block nonspecific binding sites, the plate was incubated with 2% (w/v) casein in PBS for 1 h at room temperature. The plates were incubated with the BG-biotin-conjugated scFv-SNAP fusion proteins with a fixed amount of 0.5 µg in 100 µL PBS for 1 h at room temperature, and then with horseradish peroxidase conjugated streptavidin (diluted 1:5000 in PBS). Intermediate washing steps in PBS plus 0.1% (v/v) Tween-20 (PBST) were carried out between incubations. The absorbance at 450 nm was measured with an Epoch spectrophotometer (BioTek, Bad Friedrichshall, Germany) after incubation with 3,3',5,5'- tetramethylbenzidine (TMB) substrate (Invitrogen). The reaction was stopped with 1 M HCl once a color change was observed (< 1 min). Competition ELISAs were carried out under the same conditions described above. After coating the plates with 100 ng EGFRex antigen per well and blocking, the unlabeled competitor (scFv-SNAP or mAb) was added in a five-fold molar excess to the labeled protein and incubated for 30 min. The biotin-labeled scFv-SNAP or mAb was then added in a defined amount (0.25 µg) per well and incubated for another 30 min. Detection was carried out as described above.

Surface plasmon resonance (SPR) spectroscopy The KD values of the BG-biotin-labeled scFv-SNAP fusion proteins and the parental mAbs were determined by SPR spectroscopy using a Biacore T200 instrument (Biacore™ Systems, GE Healthcare, Uppsala, Sweden). All experiments were performed at 10°C with HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) Tween-20) at a constant flow rate of 30 µL/min. For affinity measurements with the parental mAbs, a CM5-S-Series sensor chip (Biacore™ Systems) ACS Paragon Plus Environment

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was functionalized with recombinant Protein A (Sigma-Aldrich, Taufkirchen, Germany).59 Between measurements, the immobilized Protein A surface was regenerated using 30 mM HCl and buffer injections were applied for referencing. The EGFRex antigen was injected at different concentrations (120 nM, 1:2 serial dilution to 0.94 nM) at a flow rate of 30 µL/min for 150 s, and dissociation was followed for 180 s. Measurements with the BG-biotin-labeled scFv-SNAP fusion protein were carried out using the Biotin CAPture Kit (GE Healthcare, Little Chalfont, UK) which contains a Sensor Chip CAP, the Biotin-CAPture reagent and two regeneration solutions. During the coupling procedure, the Biotin CAPture-reagent was injected first and hybridized on the chip surface, and the BG-biotinlabeled scFv-SNAP fusion protein was then injected and captured. The EGFRex antigen was injected next, and the surface could then be regenerated for further experimental measurements. For regeneration the Regeneration Stock 1 and 2 were used which were part of the kit according to the manufactures instructions. The dissociation and association flow rates as well as the concentration of the EGFRex antigen were maintained as for the measurement with the mAbs described above. The binding curves were evaluated using Biacore T200 Evaluation Software (Biacore™ Systems).



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Author information *

Corresponding author

Email: [email protected] Phone: +49-241 6085-13281; Fax: +49-241 6085-10000

Notes The authors declare no competing financial interests.

Acknowledgments The authors would like to thank Magdalena Bialon for providing the EGFRex antigen and Severin Schmies for excellent technical support. We would also like to thank Dr. Richard M Twyman for critical reading of the manuscript.

Abbreviations scFv, single chain fragment variable; EGFR, epidermal growth factor receptor; mAb, monoclonal antibody; SPR, surface plasmon resonance; DTT, dithiothreitol; BG, benzylguanine; ELISA, enzyme linked immunosorbent assay; EGFRex, epidermal growth factor receptor extracellular domain; FDA, Food and Drug Administration; IMAC, immobilized metal affinity chromatography; MFI, mean fluorescence intensity; RU, response unit


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SDS-PAGE analysis of BG-fluorophore-labeled scFv1711-SNAP and scFv2112-SNAP. Lane 1: color prestained protein standard, broad range (11-254 kDa). Lanes 2 and 3: scFv1711-SNAP BG-488 and scFv2112SNAP BG-488 after staining with Coomassie Brilliant Blue. The same scFv fusion proteins shown in lane 2 and 3 were also visualized by their BG-488 fluorescence using the Cri Maestro™ imaging system with the blue filter set and are shown in lane 4 (scFv1711-SNAP BG-488) and lane 5 (scFv2112-SNAP BG-488). Dye spectra were unmixed using Cri Maestro™ software 2.2. 84x56mm (300 x 300 DPI)



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The direct and indirect measurement of scFv2112-SNAP and scFv1711-SNAP binding specificity was confirmed using different approaches. (A) First, 150 nM of scFv2112-SNAP (black curve) and scFv1711SNAP (dashed curve) labeled with BG-488 showed specific binding to the three EGFR+ cell lines, A431, MDA-MB-468 and L3.6pl. Nonspecific binding was not observed on the EGFR– cell line U937. (B) Indirect detection of scFv2112-SNAP and scFv1711-SNAP specific binding to the same cell lines using an anti-SNAP antibody and a goat anti-mouse IgG PE conjugate. (C) Binding of scFv2112-SNAP (black curve) and scFv1711-SNAP (gray curve) coupled to BG-biotin using EGFRex-coated ELISA plates. The binding of scFvSNAP BG-biotin fusion proteins to EGFRex was revealed following incubation with the peroxidase conjugated streptavidin substrate by reading the OD at 450 nm. The standard error of the means (±SD) is represented by error bars from at least three independent experiments each measured in triplicate. (D) Representative experiment showing the binding of scFv2112-SNAP BG-488 to A431 cells by confocal live cell imaging microcopy after incubation on ice for 30 min with 150 nM scFv2112-SNAP (scale bar = 10 µm). 450x343mm (300 x 300 DPI)


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Competitive inhibition of the binding of scFv2112-SNAP, scFv1711-SNAP and the parental mAbs measured by flow cytometry. EGFR+ MDA-MB-468 cells were incubated with a 10-fold molar excess of unlabeled cetuximab or panitumumab (A) and scFv2112-SNAP or scFv1711-SNAP (B), before washing and incubating the cells with a fixed concentration of BG-488-labeled scFv-SNAP (A and B). (C) The parental mAbs were also tested against each other to measure their competitive binding inhibition. The assay was also conducted using non-specific mock-scFv-SNAP (A and B) and mock-mAb (C) to evaluate the specificity of the competition. 177x157mm (300 x 300 DPI)



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Competitive ELISA using scFv2112-SNAP and scFv1711-SNAP labeled with BG-biotin. ELISA plates were coated with 100 ng/well EGFRex overnight at 4°C. After blocking with 2% (w/v) casein for 30 min, the competitors were incubated with the antigen before 0.25 µg scFv2112-SNAP BG-biotin (A) or scFv1711SNAP BG-biotin (B) was added to the wells. Bound scFv-SNAP BG-biotin fusion proteins were detected with horseradish peroxidase-conjugated streptavidin. Mean absorbance values (±SD) at 450 nm are presented from at least three independent measurements in triplicates 116x249mm (300 x 300 DPI)


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Sensograms for cetuximab (A) and panitumumab (B) measured with a Protein A Chip. Five different concentrations of EGFRex are demonstrated (30, 15, 7.5, 3.75 and 1.88 nM) with different colors and the corresponding fits are shown in black. The response units (RU) are plotted against time (s). The data were processed using Biacore T200 evaluation software. 177x144mm (300 x 300 DPI)



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Four-step procedure to measure the binding affinity of scFv-SNAP fusions using the Biotin CAPture Kit (GE Healthcare) by SPR spectroscopy. After each measurement, the sensor chip can be regenerated (step IV). (A) The Biotin CAPture reagent contains streptavidin conjugated to a single stranded DNA oligonucleotide complementary to the single-stranded DNA on the Sensor Chip CAP surface. (B) Sensograms for scFv2112SNAP. (C) Sensograms for and scFv1711-SNAP. The evaluation was processed based on the Biacore T200 evaluation software and the response units are plotted against the time in seconds. 134x189mm (300 x 300 DPI)


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Measurement of equilibrium binding constants (KD) for scFv2112-SNAP (A) and scFv1711-SNAP (B) by flow cytometry using concentrations series of each scFv. The highest MFI-value of the scFv-SNAP fusion proteins bound to EGFR+ cells (L3.6pl) was set to 100% and all other data points were normalized to this value. The experiments were carried out six times and KD values were calculated by fitting a nonlinear regression curve. 98x161mm (600 x 600 DPI)



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300x199mm (300 x 300 DPI)


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SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants

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