N-Linked Glycosylation of Antibody Fragments in Escherichia coli

Feb 14, 2011 - expression systems, but the discovery of the N-linked protein glyco- ... are amenable to bacterial N-linked glycosylation, thereby impr...
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N-Linked Glycosylation of Antibody Fragments in Escherichia coli Christian Lizak, Yao-Yun Fan, Thomas Christian Weber, and Markus Aebi* Department of Biology, Institute of Microbiology, ETH Z€urich, CH-8093 Z€urich, Switzerland

bS Supporting Information ABSTRACT: Glycosylation is the predominant protein modification to diversify the functionality of proteins. In particular, N-linked protein glycosylation can increase the biophysical and pharmacokinetic properties of therapeutic proteins. However, the major challenges in studying the consequences of protein glycosylation on a molecular level are caused by glycan heterogeneities of currently used eukaryotic expression systems, but the discovery of the N-linked protein glycosylation system in the ε-proteobacterium Campylobacter jejuni and its functional transfer to Escherichia coli opened up the possibility to produce glycoproteins in bacteria. Toward this goal, we elucidated whether antibody fragments, a potential class of therapeutic proteins, are amenable to bacterial N-linked glycosylation, thereby improving their biophysical properties. We describe a new strategy for glycoengineering and production of quantitative amounts of glycosylated scFv 3D5 at high purity. The analysis revealed the presence of a homogeneous N-glycan that significantly increased the stability and the solubility of the 3D5 antibody fragment. The process of bacterial N-linked glycosylation offers the possibility to specifically address and alter the biophysical properties of proteins.

’ INTRODUCTION Recombinant antibody fragments represent the next generation of antibody-based reagents for therapeutic and diagnostic applications.1 Because of their smaller size and their simpler architecture, they can be expressed in microbial expression systems in high yields, resulting in a fast and inexpensive production.2 Antibody fragments, like single-chain Fvs (scFvs), show better tissue penetration and better accessibility to cryptic epitopes than full-length antibodies suggesting them as candidates for the treatment of cancer, as well as immunological and infectious diseases.1 On the other hand, their small size results in a rapid renal clearance rate, and many antibody fragments are prone to instability and aggregation due to exposed hydrophobic surfaces usually capped by the Vκ or Vλ domains in full-size immunoglobulins.3,4 N-Linked glycosylation has been successfully used as a strategy to improve the pharmacokinetic and biophysical properties of proteins.5,6 Besides the well-characterized impact of the N-glycan attached to the Fc domain of IgG antibodies on Fc receptor mediated effector functions, the recognition of distinct N-glycans by carbohydrate binding proteins and lectins enables tissue- and cell-specific targeting of therapeutic proteins.7 The introduction of additional N-glycans to proteins is, on one hand, used to increase the serum half-life and consequently the biological activity, especially of protein hormones. On the other hand, it can improve the stability and solubility of proteins.8,9 However, r 2011 American Chemical Society

the major challenge of all these approaches resides in the production of a well-defined, homogeneous glycoprotein. The current production of therapeutic glycoproteins in eukaryotic expression systems, particularly in Chinese hamster ovary (CHO) cell lines, results in glycan heterogeneities that can confer significant differences to glycoprotein function.10 Alternative methods for the production of homogeneous glycoproteins include engineering of glycosylation pathways in Pichia pastoris and in vitro remodeling of glycoproteins. In the latter approach, the glycopeptide linkage is first established by chemical synthesis or by digestion of heterogeneous N-glycans using glycoside hydrolases before the glycan can be elaborated by the action of glycosyltransferases or an endo-glycoside hydrolase.11 Even though these systems are able to produce homogeneous glycoproteins, the drawback, especially of the in vitro strategies, lies in the incapability of providing quantitative amounts of glycoproteins. The discovery of the protein N-glycosylation machinery in the ε-proteobacterium Campylobacter jejuni and its successful functional transfer into Escherichia coli has raised the opportunity to produce recombinant glycoproteins in bacteria. When reconstituted in E. coli, this general N-glycosylation system assembles a Received: November 19, 2010 Revised: December 28, 2010 Published: February 14, 2011 488

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unique lipid linked heptasaccharide (GalNAc-R1,4-GalNAcR1,4-[Glc-β1,3-]GalNAc-R1,4-GalNAc-R1,4-GalNAc-R1,3-Bacβ1, where Bac is Bacillosamine) on the cytosolic side of the inner membrane. The lipid linked oligosaccharide is then translocated to the periplasmic side of the membrane, where it is transferred to asparagines of acceptor proteins that are located within an extended glycosylation consensus sequon (D/E-X-N-Z-S/T, where X, Z 6¼ P).12-14 With the present study, we wanted to elucidate whether this bacterial glycosylation system is an appropriate platform for the generation of homogeneous glycoproteins. For this purpose, we used the murine single-chain fragment of the anti-His tag antibody 3D5 as a model protein, since the 3D5 scFv is a wellcharacterized protein and it was previously engineered to obtain a highly soluble and stable protein.15 We describe a strategy for the efficient glycosylation of 3D5 resulting in pure glycoprotein. The analysis of glycosylated 3D5 shows the exclusive presence of the desired heptasachharide that did not impair the antigen binding affinity of 3D5 scFv. The quantitative production of glycosylated 3D5 allowed us to biophysically characterize the well-defined 3D5-glycoconjugate and we can show that the presence of two N-glycans on 3D5 significantly increases the solubility and the stability against proteolytic degradation.

fragments were combined in an overlap PCR with primers 1 and 11. The PCR product was digested with NsiI and SalI and ligated into the plasmid pEC(AcrA-per) digested with the same enzymes. The resulting plasmid encodes for the anti-His tag scFv 3D5 having a PelB leader sequence upstream and a Myc tag downstream to the 3D5 sequence, but has an incomplete linker sequence with an introduced NheI and FseI restriction site. Plasmid pCL15 was generated by ligation of phosphorylated, double-stranded DNA of oligonucleotides 12 and 13 into the plasmid pCL14 digested with NheI and FseI. Plasmid pCL21 was generated by ligation of phosphorylated, double-stranded DNA of oligonucleotides 14 and 15 into the plasmid pCL15 digested with NheI and AgeI. Plasmid pCL22 was generated by ligation of phosphorylated, double-stranded DNA of oligonucleotides 16 and 17 into the plasmid pCL15 digested with AgeI and FseI. Plasmid pCL23 was generated by ligation of phosphorylated, double-stranded DNA of oligonucleotides 16 and 17 into the plasmid pCL21 digested with NheI and AgeI. Plasmids pCL16 and pCL25 were constructed by cloning of the PCR fragment amplified with primers 18/19 and 20/19 respectively, using plasmid pIH1 as template. The PCR products were digested with NheI and FseI and ligated into the plasmid pCL14 digested with the same enzymes. The resulting plasmids encode for 3D5 scFv versions, where the linker sequence is replaced by a sequence of AcrA. For the construction of plasmids pCL29 and pCL30 the CmR cassette of pACYCpgl and pACYCpgl_mut was replaced with a KanR cassette by homologous recombination according to the method described by Datsenko and Wanner.16 Briefly, the KanR cassette was PCR amplified with primers 21 and 22, using plasmid pET24b as a template. The linear PCR product was transformed into E. coli XL1 Blue cells carrying the plasmid pKD20 encoding for the λ Red recombinase and plasmid pACYCpgl or plasmid pACYCpgl_mut. Transformants were selected for kanamycin resistance and analyzed for the presence of the KanR gene in the pACYC184 backbone via colony PCR. Test Expression of scFv Constructs and Immunoblot Analysis. For analytical characterization of 3D5 glycosylation, the corresponding anti-His scFv construct was co-transformed with the plasmids pACYCpgl and pACYCpgl_mut, respectively, into E. coli SCM6 cells. 5 mL precultures were inoculated from a single clone and grown overnight at 37 °C in LB medium. Main cultures were inoculated to OD600 = 0.05 in 15 mL LB medium and grown at 37 °C to OD600 = 0.5. Cultures were induced by addition of arabinose to 0.2% (w/v) and grown for 4 h at 24 °C. For extraction of periplasmic proteins, a culture volume equivalent to 3 units of OD600 was harvested by centrifugation, resuspended in 150 μL extraction buffer, consisting of 30 mM Tris-HCl (pH 8.5), 20% (w/v) sucrose, 1 mM EDTA, and 1 mg/mL lysozyme (Sigma), and incubated for 1 h at 4 °C. A final centrifugation step yielded periplasmic proteins in the supernatant. Glycosylation of anti-His scFv constructs was analyzed by SDS-PAGE (performed according to Laemmli). Immunodetection was performed with anti-c-Myc monoclonal antibody (Calbiochem) and anti-glycan serum hR6 (Amber S. and Aebi M., in preparation). Anti-rabbit IgG-HRP (Santa Cruz) and antimouse IgG-HRP (Santa Cruz) were used as secondary antibodies. Detection was carried out with ECL Western Blot Detection Reagents (GE Healthcare). Large Scale Protein Expression and Purification. For expression of (i) nonglycosylated anti-His scFv the plasmid pCL23 was transformed alone, for the expression of (ii) glycosylated anti-His scFv the plasmid pCL23 was co-transformed with the

’ EXPERIMENTAL PROCEDURES Materials. All chemicals were obtained from Fluka unless stated otherwise. Restriction enzymes were purchased from New England Biolabs and Fermentas. Oligonucleotide synthesis and DNA sequencing was performed by Microsynth AG. E. coli XL1 Blue (Stratagene) was used as a host for cloning. Adult male NMRI (Han) mice were bred and housed in the SPF rodent facility RCHCI at the ETH Zurich, Switzerland. Animal experiments were approved by the Swiss authorities, Cantonal Veterinary Department, Zurich, license no. ZH 156/2007, and performed according to the legal requirements. Strains and Growth Conditions. Strains and plasmids are listed in Supporting Information Table S1. E. coli strains were grown on LB or TB medium and kanamycin at 50 mg/L, chloramphenicol at 34 mg/L, and ampicillin at 100 mg/L were added to the media for selection as needed. Construction of Plasmids. Oligonucleotides used for cloning are listed in Supporting Information Table S2. Plasmid pCL3 was constructed by cloning of the PCR fragment amplified with primers 1 and 2, using plasmid pKB2Hmut12C30AilC30A as a template. The PCR product was digested with NsiI and SalI and ligated into the plasmid pEC(AcrA-per) digested with the same enzymes. The resulting plasmid encodes for the anti-His tag scFv 3D5 with a PelB leader sequence upstream and a Myc tag downstream to the 3D5 sequence. Plasmids pCL5 and pCL7 were constructed by QuickChange Mutagenesis (Stratagene) of pCL3 with oligonucleotides 3/4 and 5/6, respectively. Plasmid pCL6 was constructed by inverse PCR with primers 7 and 8, using pCL3 as template. The PCR product was digested with AgeI and self-ligated. Plasmids pCL5-pCL7 encode for a 3D5 version with an introduced N-glycosylation consensus sequence into the linker region between the VL and VH domain. Plasmid pCL14 was generated as an intermediate construct to provide a more general strategy for further linker derivatives. Therefore, the coding sequences of 3D5 VL and VH were PCR amplified separately with primers 1/9 and 10/11, using plasmid pKB2Hmut12C30AilC30A as template. Subsequently, both 489

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prepared in 2-fold serial dilutions from 8 μM to 1 μM, and the antibody fractions were then analyzed by surface plasmon resonance (BIAcore 3000 system) using a low-density coated chip. The CM5 chip (BIAcore) was prepared by covalently coupling recombinant 7B89-His6, at a concentration of 2.5  10-7 M. 20 μL of single chain antibody were injected using the kinject command at a flow of 20 μL/min. The binding curves were analyzed with the BIAevaluation 3.2 software, and KD values were obtained by curve fitting according to the Langmuir equation for 1:1 binding. Protein Stability. To investigate the serum stability of 3D5, human serum was isolated from 10 mL whole blood according to the method described by Harlow and Lane.18 1.5 mL of serum was spiked with 3D5-Non and 3D5-Di to 1 μM each and 20 μL samples were taken after 0, 0.5, 1.5, 4, 10, 24, and 48 h of incubation at 37 °C. After dilution with 80 μL ddH2O, samples were analyzed by SDS-PAGE and visualized by immunoblot with the anti-c-Myc monoclonal antibody. For evaluation of the scFv monodispersity, size exclusion chromatography was performed. Therefore, purified 3D5-Non and 3D5-Di protein, respectively, at a concentration of 7.0  10-6 :: M, was incubated at 37 °C for 72 h and then analyzed on an AKTA FPLC system using a Superdex 75 10/300 column (GE Healthcare). Proteolytic stability of nonglycosylated and diglycosylated anti-His scFv was tested against endoproteinase Asp-N. For this, 0.75 μg Asp-N (Calbiochem) was mixed with 3D5-Non and 3D5-Di, respectively, (at 5 μM) in a 1 mL reaction and 75 μL samples were taken after incubation of 0, 0.5, 1, 2, 3, 4, 6, and 8 h at 37 °C. To stop the reaction, samples were mixed with 4 Laemmli buffer and incubated at 96 °C for 10 min. Protein degradation was visualized by SDS-PAGE followed by immunoblot analysis. Since comparable detection of 3D5 with the secondary anit-mouse IgG-HRP alone was observed during this study, the immunoblot was incubated only with the secondary antibody, to detect potential 3D5 degradation products. Biodistribution Studies. The serum half-life and the in vivo targeting performances of non- and diglycosylated anti-His scFv were quantitatively analyzed in mice by radio-iodination. Purified 3D5-Non and 3D5-Di were radiolabeled using iodine-125 (PerkinElmer) and Chloramine-T solution (0.25 μg Chloramine-T per 1 μg protein) followed by purification on a PD-10 column (GE Healthcare) and injected into the tail vein of immunocompetent, eight-week-old NMRI mice (Taconic, 3 mice per group). 10 μL blood samples were taken from the great saphenous vein 3, 15, 45, 180, and 1440 min after injection, and mice were sacrificed subsequently. Organs were weighed and radioactivity of organs and blood samples was counted with a Cobra γ-counter (Packard). Radioactivity content of blood samples and representative organs was expressed as the percentage of the injected dose per gram of tissue (%ID/g). Protein Solubility. For solubility determination in PBS, purified native 3D5-Non and 3D5-Di proteins were concentrated to 440 μM and a stock solution of 50% (w/v) PEG 8000 was prepared in the same buffer. 20 μL of protein sample was mixed with various concentrations of PEG (11-21%) in a final volume of 40 μL and samples were incubated for 1 h at room temperature. The precipitated protein was removed by centrifugation at 16900  g (14000 rpm) in an Eppendorf tube. Supernatants were diluted 30-fold and loaded together with three standard samples of known protein concentration on the same SDS gel. Coomassie-stained gels were scanned densitometrically and the concentration of soluble protein in micromolarity was

plasmid pCL29 into E. coli SCM6 cells. 25 mL precultures in LB medium were inoculated from a single clone and grown overnight at 37 °C. Main cultures were inoculated to OD600 = 0.05 and grown in 1 L TB medium (i) or LB medium (ii) at 37 °C to OD600 = 0.5. For induction, arabinose was added to 0.2% (w/v) and cultures were grown for 4 h at 24 °C. Cells were harvested by centrifugation. For preparation of periplasmic extracts, cell pellets were resuspended to 30 units of OD600/mL in the extraction buffer described above and incubated for 2 h at 4 °C. A final centrifugation step yielded periplasmic proteins in the supernatant. For affinity purification of 3D5, the periplasmic extract was adjusted for 30 mM Mes-NaOH (pH 5.5), 500 mM NaCl, and 2 mM CaCl2 and purified as described previously.15 Briefly, a 3 mL gravity flow affinity column with a Gly-Ser-GlySer-Gly-His8-peptide coupled to NHS-Sepharose was equilibrated with 15 mL buffer A (20 mM Mes-NaOH (pH 6.5), 500 mM NaCl), before the periplasmic extract was loaded onto the column. The column was washed with 30 mL buffer A and the 3D5 protein was eluted in 6 mL gradient steps of 50 mM, 100 mM, and 300 mM imidazole in buffer B (50 mM Caps-NaOH (pH 10), 500 mM NaCl), and 1 mL elution fractions were collected. Fractions containing the anti-His scFv were identified via SDS-PAGE followed by Coomassie blue staining and were pooled. For the purification of diglycosylated 3D5 protein, pooled fractions were desalted against buffer C (50 mM MesNaOH (pH 6.5)) with a HiPrep 26/10 desalting column (GE Healthcare) and the sample was loaded onto a 1 mL ResourceQ ion exchange column (GE :: Healthcare). Different 3D5 glycoforms were eluted on an AKTA FPLC system in a linear gradient of 0% to 50% buffer D (50 mM Mes-NaOH (pH 6.5), 200 mM NaCl) in 100 column volumes and fractions containing pure diglycosylated 3D5 were pooled. For all subsequent experiments, pure non- and diglycosylated anti-His scFv were dialyzed against PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4 (pH 7.4)) and concentrated, if needed, with an Amicon Ultra centrifugal filter device (10 000 MWCO, Millipore). Concentration of 3D5 protein was determined by A280 measurements (ε = 43 110 M-1 cm-1). Mass Spectrometry Analysis. 50 μg of purified 3D5-Di in 75 μL PBS were incubated with 240 μg proteinase K (Roche) overnight at 55 °C. The proteinase K digest was loaded onto a carbon column and washed with 0.1% trifluoroacetic acid (TFA). Glycopeptides were eluted with 25% acetonitrile (ACN) in 0.1% TFA and dried down in the glass tube for permethylation. The sample was redissolved in a slurry of finely ground NaOH pellets in dimethyl sulfoxide (∼0.2 mL), followed by addition of 0.1 mL of methyl iodide. The reaction mixture was vortexed for 20 min at room temperature, quenched with 0.2 mL of water, followed by chloroform/water extraction, and permethylated N-glycans were dried under a N2-stream. MALDI-MS and MS/MS analyses of permethylated glycans were performed on a 4800 Proteomics Analyzer (Applied Biosystems), operated in the positive reflectron mode. For MS acquisition, the permethylated samples were dissolved in acetonitrile and mixed 1:1 with 2,5-dihydroxybenzoic acid (DHB) matrix (10 mg/mL in 50% acetonitrile), for spotting onto the target plate. The potential difference between the source acceleration voltage and the collision cell was set to 2 kV to obtain the high-energy CID fragmentation pattern. Other parameters and conditions were described previously.17 SPR-Spectroscopy. For affinity measurements and kinetic constant determination, purified 3D5-Non and 3D5-Di were 490

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Figure 1. Glycoengineering of the single-chain antibody fragment of 3D5. (a) The structure of 3D5 (PDB entry 1kTR) shows the arrangement of the two variable domains (VL in cyan and VH in blue) and the localization of a bound antigen (His4 peptide in red). The linker region (GGGGS)4 connecting the two variable domains is too flexible to be seen in the crystal structure but is indicated graphically. (b) Linker sequences used for the insertion and characterization of bacterial N-glycosylation consensus sequences into 3D5. Potential glycosylation sites are highlighted in yellow. Amino acids in positions -1 and þ1 of the asparagine that contribute to an improved glycosylation are marked in red. Amino acids derived from the natural C. jejuni glycoprotein AcrA are marked in green. (c-e) Periplasmic extracts of E. coli SCM6 cells transformed with either the mutated (pgl-) or the functional (pglþ) C. jejuni glycosylation machinery and expressing 3D5 variants with the indicated linker sequence. Proteins were separated by SDS-PAGE and probed with anti-c-Myc antibody or glycan-specific antiserum hR6 (Figure 1c only). The numbers of N-glycans on 3D5 are indicated at the right side of each gel.

calculated for each sample with the help of the applied standards. Measurements were performed in triplicate for both proteins. To determine the solubility limit for 3D5-Non and 3D5-Di, the logarithm of protein concentration in the supernatant was blotted as a linear function of PEG concentration and extrapolated to zero PEG concentration.

band at increased molecular weight for 3D5-GI and about 40% of the protein 3D5-GII showed a mobility shift to an increased molecular weight after SDS PAGE analysis. Both proteins reacted with the glycan specific antiserum hR6 in the presence of a competent pgl system, whereas no signal was detected for 3D3-GIII (Figure 1c). Our experiments identified the linker of the 3D5 single-chain fragment as a suitable region for the insertion of bacterial Nglycosylation sites, but indicated incomplete glycosylation. To improve the glycosylation efficiency, we designed the protein 3D5-GIV containing the optimized consensus sequence DQNAT, described by Chen et al.,21 in the middle of the linker region (Figure 1b). We also replaced the original linker region by a flexible domain of the natural C. jejuni glycoprotein AcrA that contains one glycosylation site.22 When the proteins 3D5-GIV and 3D5-AcrA-1 were expressed in glycosylation competent E. coli cells, we observed almost complete glycosylation after separation by SDS-PAGE and subsequent immunoblot analysis. For both proteins, only a population of less than 10% migrated at the original molecular weight of the nonglycosylated protein (Figure 1d). The increased occupancy of the optimized acceptor site was also reflected by the expression of constructs containing two glycosylation sites. With the construct 3D5-GII-IV carrying one original and one optimized sequon, preferentially monoglycosylated 3D5 was obtained. In contrast, the expression of the construct 3D5-GIV-IV with two optimized sequons resulted in diglycosylated protein only. For the expression of the construct 3D5-AcrA-1-2 where the AcrA sequence was N-terminally extended to display a second glycosylation site, also complete

’ RESULTS Glycoengineering of 3D5 scFv. ScFv only consist of the antigen binding domains of an IgG antibody and therefore usually do not carry N-glycans.19 In order to obtain glycosylated 3D5, we first had to introduce glycosylation sites into the 3D5 protein. As a position for the insertion, we chose the linker region connecting the variable light chain (VL) with the variable heavy chain (VH) (Figure 1a). According to the structure of 3D5, this region represents a flexible and exposed part of the protein and therefore fulfills the substrate requirements for the post-translocational glycosylation system of bacteria.20 Due to its distal location to the antigen binding region, N-glycosylation of this part of the protein should also not affect antigen binding. To determine the optimal position of a glycosylation site within the linker region, we inserted the extended bacterial consensus sequence (D/E-X-N-Z-S/T with X, Z 6¼ P)13 with a minimal number of mutations at three different positions, resulting in the constructs 3D5-GI (DGNST), 3D5-GII (DSNGT), and 3D5-GIII (DGNGS) (Figure 1b). The expression of these constructs in glycosylation competent (pglþ) E. coli SCM6 cells indicated glycosylation of 3D5-GI and 3D5-GII only. An immunoblot against the C-terminal Myc tag showed a faint 491

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Figure 2. Isolation and characterization of pure and homogeneously diglycosylated 3D5 single-chain antibody. (a) Separation of 3D5 glycoforms by anion exchange chromatography. An affinity purified mixture of 3D5 glycoforms was loaded onto a ResourceQ column and the different glycoforms were eluted in a linear gradient with NaCl. The absorption at 280 nm is indicated at the left y-axis, whereas the concentration of NaCl is shown on the right. The collected fractions were analyzed by SDS-PAGE followed by Coomassie staining, and the numbers of N-glycans on 3D5 are indicated at the right side of the gel. (b) Size exclusion chromatography and SDS-PAGE analysis of purified samples of nonglycosylated and diglycosylated single-chain antibody (3D5-Non and 3D5-Di). (c) MALDI-MS profile of permethylated glycans attached to 3D5-Di. 3D5-Di was digested with proteinase K, fractionated by a carbon column, and eluted with 25% ACN in 0.1% TFA. The eluent was permethylated, and the fully methylated glycan was shown as m/z = 2061, where incomplete methylation was detected as -14U. (d) MALDI-MS/MS sequencing of permethylated glycan attached to 3D5-Di. Precursor ion of m/z = 2061 from Figure 2c was selected for sequencing, and fragment ions were assigned according to Yu et al.28 Structural information confirmed the identity of the C. jejuni N-glycan attached to the peptide, where peptide residues after proteinase K digestion were shown as fragment ion at m/z = 362. (Red hexagon, bacillosamine; yellow squares, GalNAc; blue circles, Glc).

diglycosylation of 3D5 was achieved (Figure 1e). Consequently, the insertion of two optimized acceptor sites led to an efficient attachment of two N-glycans to the 3D5 single-chain antibody. In the following experiments, we focused on the expression of construct 3D5-GIV-IV. Production of Pure Diglycosylated 3D5. When expanding the expression of 3D5-GIV-IV to 5 L shaking flask cultures to obtain quantitative amounts of glycoprotein, we observed a reduced glycosylation level and our data suggested that loss of the pgl-encoding plasmid during cultivation was the primary cause of this effect (data not shown). Therefore, we exchanged the resistance cassette on the plasmid pACYCpgl from chloramphenicol to kanamycin. Indeed, the glycosylation efficiency increased significantly, but we still obtained a subpopulation of nonglycosylated 3D5 protein (data not shown). Therefore, we developed a glycoprotein purification strategy to isolate the diglycosylated 3D5 scFv: First, we used an immobilized His8peptide to affinity purify the different glycoforms of 3D5. When this mixture was applied to an anion exchange chromatography, different peaks at 280 nm appeared upon elution with increasing

NaCl concentration. The analysis of the chromatographic profile by SDS-PAGE revealed the separate elution of diglycosylated 3D5 (3D5-Di) from a mixture of different glycoforms (Figure 2a). Therefore, an appropriate fraction collection allowed the isolation of pure diglycosylated 3D5 protein. The purity and homogeneity of diglycosylated 3D5 protein was confirmed by SDS-PAGE and size exclusion chromatography (SEC) analysis. The defined elution of 3D5-Di in the SEC profile at the expected monomeric size showed that the attachment of two N-glycans to the linker region of 3D5 does not cause aggregation or diabody formation (Figure 2b). MS Analysis of N-Glycans Attached to 3D5. In order to elucidate the homogeneity of the attached N-glycans, we performed a MALDI-MS analysis of a proteinase K treated and subsequently permethylated sample of purified 3D5-Di. The resulting MS spectra showed a main ion at m/z = 2061, representing the fully methylated N-glycan and a second ion at m/z = 2047 resulting from partial undermethylation (Figure 2c). The MS/MS spectrum of the main ion at m/z = 2061 demonstrated a fragmentation pattern typical for the N-glycan structure 492

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Figure 3. Characterization of the impact of attached N-glycans on the properties of 3D5. (a) Human serum was spiked with 1 μM of purified 3D5-Non and 3D5-Di protein each and incubated at 37 °C. Samples were taken at indicated time points, separated by SDS-PAGE, and 3D5 serum stability was visualized by immunoblot analysis. The position of 3D5-Non (Non) and 3D5-Di (Di) is indicated at the right side of the gel. (b) Pharmacokinetic properties of 125I-labeled 3D5-Non and 3D5-Di protein were evaluated via injection into eight-week-old NMRI mice (n = 3). The remaining radioactivity in blood samples taken at different time points post injection was determined and referred to as the percentage of the injected dose per gram of blood (%ID/g). An autoradiography of 125I-labeled 3D5-Non and 3D5-Di samples after SDS-PAGE is shown in the inlet. (c) The proteolytic stability of 3D5-Non and 3D5-Di, respectively, was evaluated by incubation with the endoproteinase Asp-N at a ratio of 10:1 (scFv:proteinase) at 37 °C. Samples were taken at indicated time points and proteolytic degradation was visualized by SDS-PAGE and subsequent immunoblot analysis. (d) A PEG precipitation assay was used to analyze the solubility of the 3D5 single chain antibody. A 440 μM protein solution in PBS was mixed with increasing concentrations of a PEG 8000 solution in the same buffer to increase the protein concentration above the solubility limit. Precipitated protein was removed via centrifugation. The concentration of soluble 3D5 in the supernatant was determined by SDS-PAGE and densitometric scanning (in triplicate) and was plotted against the corresponding PEG concentration.

of C. jejuni (GalNAc-R1,4-GalNAc-R1,4-(Glc-β1,3)-GalNAcR1,4-GalNAc-R1,4-GalNAc-R1,3-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-D-Glc) (Figure 2d).23 Consequently, the expression of 3D5-GIV-IV in the engineered E. coli SCM6 cell line in combination with the developed purification technique led to 2 mg of pure and homogeneously diglycosylated 3D5 single-chain antibody per liter of culture. In comparison, the expression of nonglycosylated 3D5 single-chain antibody resulted in 8 mg protein per liter of culture (data not shown). Antigen Binding Affinity of Diglycosylated 3D5. The availability of pure diglycosylated 3D5 single-chain antibody allowed us to characterize the impact of the C. jejuni N-glycans on the properties of 3D5. First, we investigated whether the attachment of two glycans to the linker region affected the antigen binding of the 3D5 single-chain antibody. A corresponding surface plasmon resonance experiment resulted in a calculated apparent KD of 0.7 μM for 3D5-Di compared to an apparent KD of 1.7 μM for 3D5-Non against the C-terminal His6 tag of coated fibronectin (Supporting Information Figure 1). The obtained values clearly show that the attachment of two N-glycans distal to the antigen binding region of 3D5 did not impair the affinity of the single-chain antibody. Serum Stability of Diglycosylated 3D5. In a next step, we addressed the serum stability of glycosylated 3D5. We spiked a sample of human serum with 1 μM of 3D5-Non and 3D5-Di each and incubated the mixture at 37 °C. The analysis of this sample at

distinct time points by SDS-PAGE and immunoblot showed the stability of both 3D5 variants in human serum for at least 48 h (Figure 3a). Furthermore, an SEC analysis of 3D5-Non and 3D5Di after incubation in PBS at 37 °C did not show any protein aggregation after 72 h (data not shown). Pharmacokinetic Properties of Diglycosylated 3D5. Knowing that both proteins, 3D5-Non and 3D5-Di, are stable at physiological conditions, we assessed the effect of the Nglycans on the pharmacokinetic properties of 3D5. For this study, both 3D5 variants were radiolabeled with Iodine-125 (inlet Figure 3b) prior to injection into NMRI mice. The analysis of the remaining radioactivity in blood samples taken at different time points post injection showed a fast clearance rate, typical for single-chain antibody fragments (Figure 3b).3,24 After 3 h, less than 4%, and after 24 h, less than 0.5% of the injected dose per gram of blood could be detected. However, no significant difference in the clearance rate of 3D5-Non and 3D5-Di could be observed. This indicated that the C. jejuni N-glycans did not affect the clearance rate of 3D5. A biodistribution analysis revealed no enrichment in kidney, liver, lung, or spleen for neither of the two 3D5 glycoforms (Supporting Information Figure 2). Proteolytic Stability of Diglycosylated 3D5. Besides the previously investigated serum stability of 3D5, we further focused on the proteolytic stability of 3D5. The endoproteinase Asp-N cleaves a polypeptide chain specifically at aspartic acid, and 3D5 493

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bacillosamine and the first GalNAc, and it tends to interact with the residues preceding the glycosylated asparagine.22 Therefore, the negative charge of the aspartic acid in the -2 position of the asparagine is shielded upon glycosylation and the glycoprotein displays one surface charge less than the nonglycosylated counterpart. On the basis of this principle, diglycosylated 3D5 was eluted before mono- and nonglycosylated 3D5 off the column. The described effect for separation of glycoforms was further increased by incorporating an additional negatively charged amino acid at the -1 position, but this resulted in a rather inefficient glycosylation site (data not shown). The subsequent analysis of the obtained diglycosylated 3D5 revealed that the protein is pure and homogeneously decorated with two C. jejuni N-glycans. Compared to other N-glycosylation systems where glycan heterogeneities are usually a big issue,11 the chosen approach using the engineered E. coli SCM6 cell line with a deleted lipopolysaccharide biosynthesis allowed the specific attachment of the N-glycan that is encoded by the C. jejuni pgl gene cluster. This glycan homogeneity might be of particular importance for the biopharmaceutical characterization of any therapeutic protein. A more detailed characterization of diglycosylated 3D5 showed that glycosylation does not induce aggregation or diabody formation and that the antigen binding affinity is maintained. The later finding is in agreement with a previous report about glycosylated scFv produced in Pichia pastoris. An in vivo analysis for the circulating half-life in the same study revealed a 2-fold increased clearance of the glyco-scFv, most likely caused by binding to cells expressing mannose receptors.26 We did not observe any effect on the half-life of 3D5 caused by the C. jejuni N-glycan. In contrast, in vivo studies with several different therapeutic proteins, including scFv, expressed in eukaryotic cell lines resulted in increased circulation times by a factor of 2-4.5,6 More specifically, the extent of terminal R(2-6) linked sialic acid on these complex type glycans is the limiting parameter for circulation times.27 In another approach, the half-life of a scFv could be increased up to 4.9-fold by the addition of polysialic acid (PSA) to an engineered C-terminal thiol residue. The coupled PSA increased the apparent size of the scFv from 30 kDa to more than 300 kDa, indicating that the attachment of glycans can exclude proteins from renal clearance in a comparable manner as PEGylation.28 More importantly, the C. jejuni N-glycan provided a beneficial impact on the stability of the 3D5 protein: Decoration of 3D5 with N-glycans prevented proteolysis by the endoproteinase Asp-N, most likely by sterically hindering the proteinase from substrate access. Therefore, bacterial glycosylation can be an interesting tool to stabilize a protein against external proteases as well as against autolysis.8,29 The other significant observation was the increased solubility of glycosylated scFv. This finding was remarkable considering that the 3D5 protein used in the current study is already engineered for increased solubility by the introduction of nine amino acid substitutions,15 and it has a determined solubility of 130 mg/mL. Even this, for scFv untypically, high solubility limit was increased 2.5-fold by the introduction of two N-glycans. During formulation of therapeutic proteins, the solubility limit might become an issue, especially for less soluble molecules like antibody fragments. Glycosylation has been used as a strategy to increase the solubility of many proteins; however, the generality of this effect has been questioned.30 Since the solubility of any substance is determined by the interaction of the solute with the solvent and the

contains 14 potential cleavage sites. Two of these sites are located in the linker region in close proximity to the attached glycans. During incubation with Asp-N, 3D5-Di remained stable over eight hours, whereas 3D5-Non started to be degraded after three hours and was almost completely degraded after eight hours when analyzed by SDS-PAGE and immunoblot (Figure 3c). Consequently, the N-glycans seemed to hinder the protease from cleavage of 3D5. Solubility Limit of Diglycosylated 3D5. Finally, we determined whether the solubility of 3D5 increased upon glycosylation. For this, we performed a PEG precipitation assay. PEG can be used to increase the protein concentration above the solubility limit by its excluded volume effect, and there exists a linear correlation between the logarithm of protein solubility and PEG concentration.25 To apply this technique to the 3D5 protein, concentrated solutions of 3D5-Non or 3D5-Di were mixed with increasing amounts of PEG 8000. After removing precipitated protein by centrifugation, the concentration of soluble protein at different amounts of PEG was determined by SDS-PAGE and densitometric gel scanning. When the obtained concentrations were plotted as logarithm against the corresponding PEG concentrations, a 2.5-fold increased solubility for 3D5-Di was observed (Figure 3d). By extrapolating the plot to zero PEG concentration, we calculated a solubility limit of 4.3 mM for 3D5Non and of 10.8 mM for 3D5-Di. When considering the slightly higher molecular mass of 3D5-Di, this results in a solubility limit of 130 mg/mL for the nonglycosylated and of 350 mg/mL for the diglycosylated 3D5 scFv.

’ DISCUSSION We demonstrated the efficient glycosylation of a scFv protein in E. coli. ScFv are usually not glycosylated,19 but we showed that the linker region of the scFv 3D5 is an appropriate region for the insertion of glycosylation sites, when the target sequence is sufficiently spaced from the folded Ig-domains. In addition, the composition of the pentapeptide acceptor sequence was crucial for glycosylation efficiency. A study by Chen et al. on hexapeptide substrates not only revealed a clear preference for aspartic acid over glutamic acid at the -2 position of the asparagine and threonine over serine as the hydroxyamino acid at the þ2 position, but also illustrated the impact of amino acids in the -1 and þ1 positions, proposing the sequence DQNAT as the optimal acceptor site.21 Our study confirmed their observations: For the variant 3D5-GII-GIV containing a DSNGT and a DQNAT site, we mainly observed monoglycosylated 3D5. When the first site was also converted to a DQNAT site in 3D5-GIV-IV, complete diglycosylation was achieved, indicating a clear preference of the DQNAT site over DSNGT. However, glycosylation efficiency was also affected by culturing conditions: Though we obtained complete glycosylation in small shaking flasks, the transfer to larger culture volumes led to incomplete glycosylation. An exchange of the selection conditions (from Cm to Kan) improved glycosylation, suggesting that scaling up of the process can be optimized to give higher yields both for glycosylation and glycoprotein. In order to obtain the desired pure diglycosylated protein, we developed a strategy for the separation of 3D5 glycoforms. In the purification by anion exchange chromatography, we took advantage of the stronger interaction of more negatively charged proteins with the quaternary ammonium ions of the column resin. As known from structural studies, the C. jejuni N-glycan is bent due to a β(1-3) linkage between 494

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intermolecular interaction between solute molecules, N-glycans can principally influence both interactions.31 Therefore, the effect of an N-glycan on protein solubility has to be determined for every class of protein. In summary, glycosylation competent E. coli cells provide a solid platform for the quantitative production of diglycosylated scFv at high purity and homogeneity. The C. jejuni N-glycan significantly affected the biophysical properties of 3D5 as it increased the solubility and the stability against proteolytic degradation. For addressing the biopharmaceutical properties of bacterially glycosylated scFv, a comprehensive glycoengineering approach seems to be indispensable. To embed this into a rational strategy, the appropriate N-glycan can be identified using a combination of scFv glycosylation with the approach of chemoenzymatic transglycosylation.32 For the final in vivo production of the desired N-glycan, the identification of novel glycosyltransferases involved in the assembly of the glycan structure, as well as the directed evolution of enzymes involved in the process of N-glycosylation, will be crucial. For this, the recently described glycophage screening system might be exploited.33

(3) Holliger, P., and Hudson, P. J. (2005) Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23, 1126–36. (4) Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544–6. (5) Elliott, S., Lorenzini, T., Asher, S., Aoki, K., Brankow, D., Buck, L., Busse, L., Chang, D., Fuller, J., Grant, J., Hernday, N., Hokum, M., Hu, S., Knudten, A., Levin, N., Komorowski, R., Martin, F., Navarro, R., Osslund, T., Rogers, G., Rogers, N., Trail, G., and Egrie, J. (2003) Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat. Biotechnol. 21, 414–21. (6) Stork, R., Zettlitz, K. A., Muller, D., Rether, M., Hanisch, F. G., and Kontermann, R. E. (2008) N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies. J. Biol. Chem. 283, 7804–12. (7) Friedman, B., Vaddi, K., Preston, C., Mahon, E., Cataldo, J. R., and McPherson, J. M. (1999) A comparison of the pharmacological properties of carbohydrate remodeled recombinant and placental-derived beta-glucocerebrosidase: implications for clinical efficacy in treatment of Gaucher disease. Blood 93, 2807–16. (8) Bernard, B. A., Yamada, K. M., and Olden, K. (1982) Carbohydrates selectively protect a specific domain of fibronectin against proteases. J. Biol. Chem. 257, 8549–54. (9) Runkel, L., Meier, W., Pepinsky, R. B., Karpusas, M., Whitty, A., Kimball, K., Brickelmaier, M., Muldowney, C., Jones, W., and Goelz, S. E. (1998) Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (IFN-beta). Pharm. Res. 15, 641–9. (10) Sethuraman, N., and Stadheim, T. A. (2006) Challenges in therapeutic glycoprotein production. Curr. Opin. Biotechnol. 17, 341–6. (11) Rich, J. R., and Withers, S. G. (2009) Emerging methods for the production of homogeneous human glycoproteins. Nat. Chem. Biol. 5, 206–15. (12) Alaimo, C., Catrein, I., Morf, L., Marolda, C. L., Callewaert, N., Valvano, M. A., Feldman, M. F., and Aebi, M. (2006) Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides. EMBO J. 25, 967–76. (13) Kowarik, M., Young, N. M., Numao, S., Schulz, B. L., Hug, I., Callewaert, N., Mills, D. C., Watson, D. C., Hernandez, M., Kelly, J. F., Wacker, M., and Aebi, M. (2006) Definition of the bacterial N-glycosylation site consensus sequence. EMBO J. 25, 1957–66. (14) Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wren, B. W., and Aebi, M. (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790–3. (15) Kaufmann, M., Lindner, P., Honegger, A., Blank, K., Tschopp, M., Capitani, G., Pluckthun, A., and Grutter, M. G. (2002) Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen. J. Mol. Biol. 318, 135–47. (16) Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97, 6640–5. (17) Yu, S. Y., Wu, S. W., and Khoo, K. H. (2006) Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans. Glycoconj. J. 23, 355–69. (18) Harlow, E., and Lane, D. (1988) Antibodies: a laboratory manual, p 119, Chapter 5, Coldspring Harbor Laboratory Press, Cold Spring Harbor, NY. (19) Huhn, C., Selman, M. H., Ruhaak, L. R., Deelder, A. M., and Wuhrer, M. (2009) IgG glycosylation analysis. Proteomics 9, 882–913. (20) Kowarik, M., Numao, S., Feldman, M. F., Schulz, B. L., Callewaert, N., Kiermaier, E., Catrein, I., and Aebi, M. (2006) N-linked glycosylation of folded proteins by the bacterial oligosaccharyltransferase. Science 314, 1148–50. (21) Chen, M. M., Glover, K. J., and Imperiali, B. (2007) From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni. Biochemistry 46, 5579–85.

’ ASSOCIATED CONTENT

bS

Supporting Information. Tables for strains and plasmids as well as for oligonucleotides, figure for affinity analysis of 3D5-Non and 3D5-Di by SPR spectroscopy, figure for biodistribution analysis of 3D5-Non and 3D5-Di in mice. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Correspondence should be addressed to Markus Aebi, Institute of Microbiology, Department of Biology, ETH Z€urich, Wolfgang-Pauli-Str.10, 8093 Z€urich, Switzerland. E-mail: aebi@ micro.biol.ethz.ch. Tel: þ41-44-632 64 13. Fax: þ41-44-632 13 75. Author Contributions

C.L., designing and performing of experiments and writing the manuscript; Y-Y.F., MS analysis of glycosylated 3D5; T.C.W., performing of mouse experiments; M.A., supervising the research and writing the manuscript.

’ ACKNOWLEDGMENT We thank the members of the Aebi lab for fruitful discussions and Flavio Schwarz and Alexander Frey for critically reading the manuscript. We are grateful to Dario Neri and Alessandro Palumbo for their help and advice with the mouse experiments and the SPR spectroscopy. We thank Miguel Valvano and Cristina Marolda for the kind gift of E. coli SCM6 and Frank Striebel for initial help with the ion exchange chromatography. This work was supported by the Swiss National Science Foundation (SNF grant 31003A_127098 to M.A.). C.L. and Y.-Y.F. are students of the Life Science Zurich Graduate School. ’ REFERENCES (1) Nelson, A. L., and Reichert, J. M. (2009) Development trends for therapeutic antibody fragments. Nat. Biotechnol. 27, 331–7. (2) Schmidt, F. R. (2004) Recombinant expression systems in the pharmaceutical industry. Appl. Microbiol. Biotechnol. 65, 363–72. 495

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(22) Slynko, V., Schubert, M., Numao, S., Kowarik, M., Aebi, M., and Allain, F. H. (2009) NMR structure determination of a segmentally labeled glycoprotein using in vitro glycosylation. J. Am. Chem. Soc. 131, 1274–81. (23) Young, N. M., Brisson, J. R., Kelly, J., Watson, D. C., Tessier, L., Lanthier, P. H., Jarrell, H. C., Cadotte, N., St Michael, F., Aberg, E., and Szymanski, C. M. (2002) Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277, 42530–9. (24) Kipriyanov, S. M., Moldenhauer, G., Schuhmacher, J., Cochlovius, B., Von der Lieth, C. W., Matys, E. R., and Little, M. (1999) Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J. Mol. Biol. 293, 41–56. (25) Middaugh, C. R., Tisel, W. A., Haire, R. N., and Rosenberg, A. (1979) Determination of the apparent thermodynamic activities of saturated protein solutions. J. Biol. Chem. 254, 367–70. (26) Wang, M., Lee, L. S., Nepomich, A., Yang, J. D., Conover, C., Whitlow, M., and Filpula, D. (1998) Single-chain Fv with manifold N-glycans as bifunctional scaffolds for immunomolecules. Protein Eng. 11, 1277–83. (27) Kronman, C., Chitlaru, T., Elhanany, E., Velan, B., and Shafferman, A. (2000) Hierarchy of post-translational modifications involved in the circulatory longevity of glycoproteins. Demonstration of concerted contributions of glycan sialylation and subunit assembly to the pharmacokinetic behavior of bovine acetylcholinesterase. J. Biol. Chem. 275, 29488–502. (28) Constantinou, A., Epenetos, A. A., Hreczuk-Hirst, D., Jain, S., Wright, M., Chester, K. A., and Deonarain, M. P. (2009) Site-specific polysialylation of an antitumor single-chain Fv fragment. Bioconjugate Chem. (29) Russell, D., Oldham, N. J., and Davis, B. G. (2009) Site-selective chemical protein glycosylation protects from autolysis and proteolytic degradation. Carbohydr. Res. (30) Sola, R. J., and Griebenow, K. (2009) Effects of glycosylation on the stability of protein pharmaceuticals. J. Pharm. Sci. 98, 1223–45. (31) Lawson, E. Q., Hedlund, B. E., Ericson, M. E., Mood, D. A., Litman, G. W., and Middaugh, R. (1983) Effect of carbohydrate on protein solubility. Arch. Biochem. Biophys. 220, 572–5. (32) Schwarz, F., Huang, W., Li, C., Schulz, B. L., Lizak, C., Palumbo, A., Numao, S., Neri, D., Aebi, M., and Wang, L. X. A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation. Nat. Chem. Biol. 6, 264-6. (33) Durr, C., Nothaft, H., Lizak, C., Glockshuber, R., and Aebi, M. (2010) The Escherichia coli glycophage display system. Glycobiology 20, 1366–72.

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