Tailoring structure–function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation

Karen Yang, Amartya Basu, Maoliang Wang, Ramesh Chintala, Ming-Ching Hsieh, Sam Liu, Jack Hua, Zhenfan Zhang, John Zhou, Mark Li, Hnin Phyu, Gerald Petti, Magda Mendez, Haleema Janjua, Ping Peng, Clifford Longley, Virna Borowski, Mary Mehlig and David Filpula1

Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, NJ 08854-3969, USA

1 To whom correspondence should be addressed. e-mail: david.filpula{at}enzon.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-{alpha} scFv at the C-terminus or within the linker segments of both scFv orientations, VL–linker–VH and VH–linker–VL. High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide–PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Indepen dent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies.

Keywords: Pichia/polyethylene glycol/therapeutic antibodies/TNF-{alpha}


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Single-chain antibodies (scFv proteins) consign antigen- binding sites within a single gene, single polypeptide design and are well established as a discovery format of choice in the disciplines of antibody and protein engineering (Begent et al., 1996Go; Dall’Acqua and Carter, 1998Go; Filpula and McGuire, 1999Go). Generation of optimal target-binding scFv proteins from phage display, yeast display or ribosome display technologies is widely utilized (Marks and Marks, 1996Go; Hanes et al., 1998Go; Hudson, 1998Go; Feldhaus et al., 2003Go). However, owing to the rapid renal clearance of scFv proteins in vivo, these minimal antigen-binding proteins are typically converted to full-length IgG antibodies before development as clinically useful antibody therapeutics, unless neither extended in vivo action nor Fc-related effector functions are required (Larson et al. 1997Go; Fitch et al., 1999Go). It has long been the goal of antibody engineers and protein scientists to develop a drug delivery method for high-affinity single-chain antibodies and other small target-binding proteins that would allowed tailored circulating lives of these simple protein scaffolds. PEGylation, the covalent attachment of polyethylene glycol (PEG) polymers to compounds, has become one of the best validated drug delivery methods for extension of serum half-life (Bailon et al., 2001Go; Greenwald et al., 2003Go) and at least six PEGylated protein therapeutics are now on the market (Harris and Chess, 2003Go). However, conjugates composed of PEG and target-binding protein ligands frequently exhibit reduction or loss of bioactivity and demonstrate substantial product heterogeneity. Some success in ligand and antibody fragment PEGylations has been reported (Clark et al., 1996Go; Pettit et al. 1997Go; Wang et al., 1998Go; Chapman et al., 1999Go; Lee et al., 1999Go; Peters and Sikorski, 1999Go; Tsutsumi et al., 2000Go), but a general strategy for creating tailored site-specific PEGylated single-chain antibodies has not been developed. An antigen-binding site may constitute about one-third of the Fv surface area; consequently, extensive random conjugations may be inactivating either through direct attachments to antigen-binding contact residues or as a result of transient steric hindrance and diffusional constraints from the long polymer strands. Since typical VL and VH domains each possess a fully buried single disulfide linkage (Padlan, 1994Go), but no free cysteines, we have designed and investigated scFv formats that permit single site-specific conjugation of maleimide–PEG polymers to single engineered cysteine sites introduced on the protein surface. In order to validate this protein engineering approach, we re-engineered a well characterized monoclonal antibody – D2E7/Humira recognizing TNF-{alpha} (Kempeni, 2000Go; Santora et al., 2001Go) – as designed scFv proteins that were expressed and purified from Pichia pastoris. Optimization of conjugate preparation with 5, 20 and 40 kDa maleimide–PEG polymers was achieved and a comparison of the physicochemical and functional properties of the purified scFv proteins and their mono-PEGylated counterparts was performed that verified their composition and maintenance of functional activity dependent on microenvironment. Pharmacokinetic studies of the conjugates in mice demonstrated that the circulating lives of the PEGylated scFv proteins could be tailored. These results demonstrate a practical and general approach for designing scFv proteins as therapeutics, without restriction to the confinements of the archetypical IgG format for clinical antibodies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Oligonucleotides were purchased from MWG Biotech (High Point, NC). Invitrogen (Carlsbad, CA) supplied P.pastoris hosts and vectors and pre-cast polyacrylamide (4–20%) slab gels. Anti-EN446 scFv and anti-218-linker rabbit serum were produced by BioSynthesis (Lewisville, TX). Streptavidin-peroxidase was obtained from Sigma (St. Louis, MO) and TMB peroxidase substrate from Moss (Pasadena, CA). TNF-{alpha} was purchased from Chemicon (Temecula, CA). Titrisol iodine solution was obtained from EM Science (Gibbstown, NJ). HiPrep 26/10 and PD-10 desalting columns (Pharmacia Biotech, Piscataway, NJ) and Poros 50 Micron HS media (Applied Biosystems, Foster City, CA) were used. mPEG–maleimide compounds were purchased from Nektar Therapeutics (San Carlos, CA; formerly Shearwater Corp.) or synthesized at Enzon Pharmaceuticals. N-Ethylmaleimide and 6-(biotinamidocaproylamido)caproic acid N-hydroxysuccinimide ester were purchased from Sigma. rProtein A Sepharose Fast Flow was obtained from Amersham BioSciences (Piscataway, NJ). Ultralink Iodoacetyl was obtained from Pierce Biotechnology (Rockford, IL). Fluorokine was purchased from R&D Systems (Minneapolis, MN). Streptavidin–phycoerythrin was obtained from BD Sciences (San Jose, CA).

scFv gene constructions

The genes for D2E7/VL–218–VH and D2E7/VH–(GGGGS)3–VL scFv were synthesized from the published sequence (US Patent 6 258 562) using peptide linkers 218 (Filpula et al., 1996Go) and (GGGGS)3 (Huston et al., 1988Go).

Expression of scFv in Pichia pastoris

P.pastoris was employed for production of the scFv variant proteins. The signal sequence from the yeast alpha mating factor was inserted directly in front of the mature coding sequence for each of the scFv proteins. The amino acid sequence of this signal peptide is Met–Arg–Phe–Pro–Ser– Ile–Phe–Thr–Ala–Val–Leu–Phe–Ala–Ala–Ser–Ser–Ala–Leu–Ala{wedge}Ala, where ‘{wedge}’ indicates the cleavage site. The 20th amino acid following the signal (the alanine after {wedge}) was also included in these constructs to facilitate efficient processing. N-terminal protein sequencing with a PROCISE protein sequencer (ABI, Foster City, CA) confirmed this correctly processed N-terminal alanine preceding the mature scFv protein sequences. The scFv genes were individually ligated at the EcoRI site into the Pichia transfer plasmid pHIL-D2 and transformed into the yeast P.pastoris host GS-115. Culture in BMGY and BMMY has been described (Wang et al., 1998Go).

Fermentation and scFv protein purification

High-density fermentations of scFv clones were performed in BMGY medium, using BioFlow IV automatic feed control fermenters (New Brunswick Scientific, Edison, NJ). Feeding of each component was optimized with respect to the dissolved oxygen level, which was set at 30%. The growth temperature was set at 29°C and pH was maintained at 6.0 using ammonium hydroxide and phosphoric acid during the run.

The scFv proteins, EN446, EN450, EN452, EN456, EN458 and EN460, were purified to 96–99% purity, as a composition of monomers and disulfide linked dimers, from the fermentation supernatant using a combination of ion-exchange and affinity column chromatography. The diafiltered supernatant was processed using DEAE column chromatography (Amersham BioSciences) and Ni-NTA resin (Qiagen, Valencia, CA). Purification of scFv proteins lacking the six-histidine-tag was also performed with ion-exchange POROS 50 HS (Applied BioSystems) or Q-Sepharose FF (Amersham BioSciences) and protein L (CBD Technologies, Buffalo, NY) affinity chromatography methods.

Maleimide–PEG reactions

Functional group analysis was conducted in two steps: reaction of the MAL–PEG with excess cysteine and determination of unreacted cysteine by titration with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (Creighton, 1989Go). Determination of active MAL–PEG was conducted at a reaction molar ratio of 1:3 (MAL–PEG:cysteine) in 50 mM sodium phosphate, pH 6.0, 1 mM EDTA. For cysteine titration by DTNB, the absorbance at 412 nm was recorded after 5 min at 25°C using 13 300 l/mol.cm as the extinction coefficient of DTNB.

Reduction and PEGylation

The free cysteine residue at the C-terminus or linker of the scFv proteins was reduced prior to reaction with MAL–PEG. The reduction solution contained 3 mg/ml scFv, 2 mM dithiothreitol (DTT), 2 mM EDTA, 100 mM sodium phosphate, pH 7.8. The reduction was conducted at 37°C for 2 h. Free DTT was removed on a HiPrep or PD-10 desalting column. The column was equilibrated with 100 mM sodium phosphate, pH 6.0, 2 mM EDTA. Near quantitative reduction of one thiol per scFv molecule was achieved as measured by DTNB assays (Creighton, 1989Go).

The typical PEGylation reaction buffer contained 1 mg/ml reduced scFv protein, 100 mM sodium phosphate, pH 6.0, 2 mM EDTA and PEG–maleimide compound at a reaction molar ratio of 10:1 (PEG:scFv). The reaction was conducted at 25°C under nitrogen for 2 h. The typical conjugation yield, as analyzed by SDS–PAGE, was about 80%. The reaction pH range 5–8 was investigated. Unreacted scFv protein could be successfully reprocessed in a second reduction and conjugation. The yield from the second conjugation reaction was similar to that obtained for the initial scFv PEGylation.

PEG–scFv conjugate purification

HS chromatography was used for purification of PEG–scFv from native scFv, high molecular weight impurities and unreacted free PEG. The column equilibration buffer contained 10 mM sodium phosphate, pH 5.0, and the gradient elution buffer was 10 mM sodium phosphate, pH 5.0, 1 M NaCl. Endotoxin present in scFv samples was removed by anion- or cation-exchange columns.

Analytical characterization of scFv and PEG–scFv

Protein concentrations were determined by measuring the UV absorbance at 280 nm, with an scFv extinction coefficient of 1.24 ml/mg.cm. The concentration was also confirmed by the bicinchoninic acid assay (BCA), obtained as a Micro BCA Protein Assay Reagent kit from Pierce Biotechnology using lysozyme or a Fab as standards.

Western blot analysis was performed with either anti-EN446 scFv or anti-218 linker rabbit antiserum as a primary antibody and goat anti-rabbit HRP as a secondary antibody. Binding was measured with a TMBM peroxidase substrate.

Iodine staining of SDS–PAGE gels was performed after the gels had been rinsed with distilled water and placed in 5% barium chloride solution. After 10 min of gentle mixing, the gels were again rinsed with water and placed in 0.1 M Titrisol iodine solution for color development (Kurfurst, 1992Go).

Mass values of scFv and PEG–scFv conjugates were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker Daltronics OmniFlex NT) using an internal standard with a similar molecular weight. Apparent molecular weights (Stoke radius) of the scFv proteins were estimated using Superdex 200 HR 10/30 gel filtration column chromatography (Amersham BioSciences) in 50 mM sodium phosphate, pH 6.5, 150 mM NaCl, with BioRad (Hercules, CA) molecular weight standards. Analysis of molecular masses on 4–20% SDS–PAGE gels was performed using protein and PEG–protein standards.

Peptide mapping was performed by published procedures (Carrey, 1989Go). The PEG–scFv (0.2 mg) was denatured and reduced in 6 M guanidine.HCl, 1 mM EDTA, 5 mM DTT, then alkylated with iodoacetamide, prior to digestion with TPCK-treated trypsin. The trypsin-generated peptide mixture was fractionated by size-exclusion chromatography (SEC) (Superdex 75) with HPLC-grade water and analyzed by SDS–PAGE and iodine staining. The unique iodine-stained fraction was subjected to protein sequencing analysis (PROCISE, Applied Biosystems).

Flow cytometry

Flow cytometry studies with WEHI-13VAR cell lines were used to analyze TNF-{alpha} binding to the cell receptors in the presence of scFv or PEG–scFv. Biotin labeled TNF-{alpha} (0.04 µg) was preincubated with the scFv or PEG–scFv (1–4 µg) in 50 µl of FACS buffer (1% FBS, 0.05% NaN3 in PBS) at 25°C for 30 min and then at 4°C for 15 min with gentle agitation. Control compounds included biotinylated soybean trypsin inhibitor (0.05 µg), polyclonal goat IgG anti-human TNF-{alpha} antibody (20 µg) and CC49 scFv (4 µg). To each 50 µl mixture of TNF-{alpha} and PEG–scFv were added 50 µl of 1x105 WEHI-13VAR cells. After 60 min of incubation at 4°C in the dark, the cells were pelleted and resuspended in 80 µl of cold FACS buffer. Streptavidin–phycoerythrin (10 µl) was added and the mixture was incubated in the dark at 4°C for 30 min. The cells were then washed twice with 1 ml of cold FACS buffer and resuspended in 0.3 ml of FACS wash buffer for analysis on FACS Calibur (BD Biosciences, San Jose, CA).

Surface plasmon resonance analysis of TNF-{alpha} interaction with scFv compounds

Surface plasmon resonance (SPR) techniques were performed using a Biacore X instrument (Biacore, Piscataway, NJ). Recombinant human TNF-{alpha} of >97% purity (Pierce) was immobilized on a CM5 chip (Biacore, Cat. No. BR-1000-14) as a 10 µg/ml solution at pH 5.0 (acetate buffer, Biacore, Cat. No. BR-1003-51). The immobilized surface was washed three times with acetate buffer, pH 4.5 (Biacore, Cat. No. BR-1003-50) and subjected to ligand stability analysis for six cycles with 500 nM native scFv. The scFv or PEG–scFv proteins served as analyte, with acetate (pH 4.5) as the regeneration buffer. Over the stable TNF-{alpha}-bound surface, different concentrations of scFv or PEG–scFv were examined for association (3 min) and dissociation (2 or 5 min) and the data were analyzed for kinetic and affinity parameters (kon, koff, KA and KD) using BiaEvaluation software (version 3.0). HBS-N (Biacore, Cat. No. BR-1003-69) was used as the running buffer in this protocol.

Neutralization of TNF-{alpha} cellular cytotoxicity by scFv compounds

WEHI-13VAR cells (ATCC No. CRL-2148), which are sensitive to TNF-{alpha} in the presence of 500 ng/ml actinomycin D, were seeded in a 96-well plate, 10 000 cells per well and incubated overnight at 37°C in a humidified incubator with 5% CO2. Propagation was on RPMI 1640 medium with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 10% FBS. A range of concentrations of scFv proteins and their PEGylated forms were added to the seeded cells in serial dilutions from 10 µg/ml to 2.5 ng/ml diluted in culture medium. Immediately following the addition of scFv compounds, rhTNF-{alpha} (Pierce) was added to each well at a concentration of 1.0 ng/ml. The cells were then allowed to grow for 24 h and cell viability was determined by addition of 15 µl of MTT (Promega, Madison, WI). The plate was read at 570 and 630 nm in a 96-well plate reader (Molecular Devices, Sunnyvale, CA). The analysis of cell rescue was performed by comparing the viability of scFv-treated cells with untreated cells in the presence of TNF-{alpha}. Cells treated with TNF-{alpha} alone exhibited a complete loss of viability.

Purification of anti-EN446 scFv polyclonal antibody

Anti-EN446 scFv antibodies raised in rabbits were purified by protein A chromatography (Amersham BioSciences) and EN450 scFv-conjugated affinity column chromatography prepared by a coupling reaction of Ultralink Iodoacetyl with the free cysteine residue of the scFv protein.

ELISA for scFv quantitation

The linear range of scFv tested was between 0.2 and 30 ng/ml. A standard curve (point to point, exponential or quadratic) for PEG–scFv compounds was fitted to 1–100 ng/ml protein concentrations and an optical reading within the linear range was used for analysis. A sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine plasma concentrations of scFv and PEG–scFv conjugates. The capture antibody was polyclonal anti-EN446 scFv antibody which was purified by protein A and EN450 scFv-conjugated affinity columns. The primary and secondary reagents were biotinylated anti-EN446 scFv antibody and streptavidin-peroxidase, respectively. Standard curves demonstrated a correlation coefficient of 0.99 or better.

Pharmacokinetics of scFv and PEG–scFv

ICR mice (female, 7–8 weeks) were supplied by Harlan Sprague Dawley (Madison, WI). Mice (3–6 per group) received an i.v. bolus via the lateral tail vein or were injected subcutaneously intrascapular, with 180–200 µl (4.3 mg scFv equivalents/kg) per mouse of the scFv and PEG–scFv compounds. Following sedation with 0.09% avertin, sampling of blood was undertaken via the retro-orbital sinus into vials containing EDTA. At 2, 15, 30 and 60 min the mice were bled 100 µl and at 3, 6, 24, 48, 72 and 96 h the mice were terminally bled by cardiac puncture. The plasma was collected following centrifugation of the blood at 5000 r.p.m. at 4°C for 5 min and immediately frozen on dry-ice. The concentration of scFv compounds was determined by ELISA. The data were modeled using WinNonlin software (WinNonLin Pharsight, Mountain View, CA) to determine pharmacokinetic parameters using a two-compartment, bolus, first-order elimination model for the intravenous samples. The correlation between observed and predicted model time point values gave coefficients of determination (r2) of >0.99.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Design strategy for monoPEGylated scFv

A structural view of VL–linker–VH architecture (Wang et al., 1998Go) is shown in Figure 1A and B. An anti-TNF-{alpha} scFv protein, derived from the clinically investigated D2E7 monoclonal antibody (Kempeni, 2000Go), was chosen as a model single-chain Fv for our investigations of site-specific PEGylations of scFv variant proteins at an engineered free cysteine residue. In order to minimize the potential for PEG polymer strands to sterically block the antigen-binding site, our preferred attachment sites were placed at the VH or VL C-terminus (with or without a C-terminal oligo-histidine tag) or the second amino acid position of the alternate peptide linkers, 218 and (GGGGS)3, that connect the VL and VH domains in either orientation. Table I summarizes the designs of the 10 scFv variant proteins in this investigation.




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Fig. 1. Two views of the VL–linker–VH anti-fluorescein 4-4-20/212 scFv model based on the Fab crystal structure. The VL is shown in red, the VH in blue, the antigen in green and the linker in yellow; position 2 of the linker is the second position from the VL terminus. A pentahistidine C-terminal tag is highlighted in turquoise bound with a nickel ion in orange. (A) Antigen-binding site is at right in the {alpha}-carbon tracing model. (B) The molecule shown in (A) is rotated 90° on two axes placing the antigen-binding site in the center of the scFv model.

 

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Table I. Anti-TNF-{alpha} scFv designs
 
Pichia expression and purification of scFv proteins

The scFv proteins, described in Table I, were all expressed by secretion from the yeast P.pastoris. Periplasmic or cytoplasmic expression in Escherichia coli of scFv proteins bearing free thiols has been problematic owing to reduced yields and aggregation (McCartney et al., 1994Go; Schmiedl et al., 2000Go). In contrast, all scFv proteins that we examined were secreted in high yield (50–100 mg/l) from P.pastoris, with no diminution of yield for scFv with free thiols. Exceptions were the two-thiol variant clones, EN458 and EN466, which secreted 5–10-fold less scFv protein. Affinity and cation-exchange chromatography were used to purify the scFv proteins to 96–99% purity with low endotoxin levels (<1 EU/ml). A notable feature of these scFv variant proteins is the presence of monomers and disulfide-linked dimers in the final preparation. These dimers are quantitatively converted to monomers by reduction and alkylation as examined by western blotting, SEC and mass spectrometric analysis. This anti-TNF-{alpha} scFv protein does not appear to have a high propensity to form non-covalent associations (Whitlow et al., 1994Go), unless constructed with a short linker (EN470), but the covalently linked surface disulfides of the dimers must be fully reduced prior to conjugation reactions, while maintaining the integrity of the two buried disulfides of the scFv. Analyses by MALDI-TOF-MS, N-terminal protein sequencing, SDS–PAGE, western blotting, SEC, DTNB and bioactivities confirmed the identity of each of the purified scFv proteins, together with similar characterizations of their corresponding bioconjugates. Representative data are summarized for EN450 versions in Table II.


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Table II. Molecular mass of EN450 scFv and PEG–scFv
 
PEG–maleimide reactions

Our initial investigations focused on validation of specific reactivity of PEG–maleimide (PEG–MAL) with only free cysteines and the stability of these activated polymers in solution. As shown in Figure 2A, maleimide undergoes alkylation reactions with sulfhydryl groups to form stable thioether bonds. The availability of synthetic PEG polymers having one maleimide group at a single terminus, shown in Figure 2B, allows thiol-directed PEGylations. Using the defined reaction parameters described in Materials and methods, we confirmed the rapid and specific reaction with cysteine, but essentially no reactivity with lysine or histidine at pH 6.0, as shown in Figure 2C. We further established the aqueous stability of maleimide in PEG–MAL polymers for optimization of reaction with scFv-thiol proteins (Figure 2D). The PEG–MAL polymer maleimide moiety displays stability over 24 h at 4°C, pH 5–7 and over 2 h at 25°C, pH 5–7, whereas, under these conditions, quantitative reaction with cysteine is completed within 5 min.



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Fig. 2. PEG–maleimide polymers. (A) Formation of thioether bond between maleimide and cysteine of scFv. (B) Structure of PEG–MAL polymers used in these studies. mPEG is monomethoxy-PEG. (C) Reaction of PEG–MAL with cysteine in 100 mM sodium phosphate, pH 6.0, 1 mM EDTA; absorbance scanned after 30 min at 25°C. Curve 1, 1 mM 20 kDa PEG–MAL; curve 2, 3 mM cysteine; curve 3, 1 mM PEG–MAL plus 3 mM cysteine. (D) Stability at 25°C of 20 kDa PEG–MAL (1 mM) in 50 mM sodium phosphate, 1 mM EDTA, pH 7.0. Absorbance (240–400 nm) was monitored at 0 (open squares), 2, 4, 22 and 33 h (top to bottom). At 33 h, 3 mM cysteine was incubated for 5 min prior to scan (closed squares).

 
scFv PEGylation

Our site-specific conjugation strategy presents an interesting circumstance. Total reduction of the engineered free cysteine is desired, while internal VL and VH disulfides must remain oxidized. An investigation of several reductants and reducing parameters demonstrated that 0.5–2 mM DTT reduction for 2 h at 37°C followed by rapid separation on desalting columns prior to conjugation was successful for near quantitative reduction of one free thiol per scFv. As determined by DTNB assays, before and after reduction, the internal disulfides were not detectably reduced.

PEG–MAL linear polymers of 5 and 20 kDa mass, plus a branched PEG–MAL polymer of 40 kDa mass, were investigated in scFv conjugations. After the conjugation yield had been optimized, the PEGylated scFv proteins were purified by cation-exchange chromatography to remove free PEG and unmodified scFv. All PEG–scFv compounds examined in further structural and functional studies contained >99% mono-PEGylated scFv. Figure 3A and B present an example of EN450 scFv conjugates analyzed on Coomassie Blue- and iodine-stained SDS–PAGE gels.






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Fig. 3. Characterization of PEG–scFv compounds. SDS–PAGE analysis of EN456 scFv and PEG–scFv conjugates. The samples were denatured by heating at 85°C for 3 min in the presence of 12 mM ß-mercaptoethanol. Protein was identified by Coomassie Blue staining (A) and PEG compounds by iodine staining (B). (A) Lane 1, molecular weight markers (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4 and 6 kDa); lane 2, EN456 scFv; lane 3, 20 kDa PEG–scFv; and lane 4, 40 kDa PEG–scFv. (B): lane 1, molecular weight markers (250, 148, 98, 64, 50, 36, 22, 16, 6 and 4 kDa); lane 2, EN456 scFv; lane 3, 20 kDa PEG–scFv; and lane 4, 40 kDa PEG–scFv. (C) Biacore binding kinetics for EN450 scFv, where scFv concentrations, 1–5, are 135, 67.5, 33.8, 16.9 and 8.4 nM or (D) for EN450 (40 kDa)–PEG–scFv, 1–5, at 3141, 1570, 785, 393 and 196 nM. (E) Rescue of WEHI-13VAR cells by EN450 scFv, N-ethyl-scFv and PEG–scFv from TNF-{alpha} (1 ng/ml) cytotoxicity.

 
Mass spectrometry, peptide mapping and SEC

MALDI-TOF-MS confirmed the predicted molecular mass of scFv and PEG–scFv compounds (Table II). Peptide mapping studies, which were undertaken to analyze the site(s) of polymer attachment to the protein, confirmed the expected single linkage of PEG to the engineered free cysteine. For example, EN456 scFv contains one free sulfhydryl at position 2 of the linker. Following trypsin digestion of 40 kDa PEG–scFv, the unique PEG–peptide was isolated by SEC and reversed-phase high-performance liquid chromatography (RP-HPLC) and subjected to protein sequence analysis. The sequence G–TSGSGKPG corresponds to a predicted linker-derived peptide, where the blank position in the sequence (–) represents the modified cysteine residue.

Gel filtration analysis of the PEG–scFv compounds (Table II) highlights a remarkable property of polyethylene glycol conjugates. The Stokes radius of PEG–scFv compounds extends far in excess of added molecular mass, resulting in a predicted molecular weight for EN450 40 kDa PEG–scFv 9-fold greater than determined by mass spectrometry. This exhibition of hydrodynamic volume expansion in PEG–scFv by the mobile and hydrophilic PEG polymer possibly plays a central role in the acquisition of altered functional properties as we describe below.

Affinity and kinetic analysis by surface plasmon resonance

Determinations of KD, kon and koff were performed by a novel Biacore analysis, wherein the ligand, huTNF-{alpha}, was immobilized directly on CM5 chips, rather than using the more conventional sandwich designs. Representative binding kinetics with EN450 scFv and 40 kDa PEG–scFv are shown in Figure 3C and D. Table III compiles kinetic values, done in triplicate, for a series of three scFv variant proteins and their mono-PEGylated counterparts. EN446 parent scFv and EN450 scFv proteins displayed similar binding kinetics. Within a common scFv mutein group, such as EN450 scFv, the effects of PEGylation are incremental, but may plateau after about 20 kDa polymer mass. Modification of the engineered VL–218–VH–his6 C-terminal cysteine with an ethyl-MAL or 5 kDa PEG–MAL has modest effects, whereas conjugates with either 20 or 40 kDa PEG display about two-log reductions in association kinetics, and the dissociation rate is nearly equivalent to the unmodified scFv. Examination of a VL–218–VH scFv variant PEGylated on position 2 of the linker, such as EN456 (data not shown), drew similar conclusions, with a nearly two-log reduction in kon but 4-fold slower koff values. When the VH–(GGGGS)3–VL–his6 scFv proteins were PEGylated with 40 kDa MAL on either the C-terminus (EN452) or linker position 2 (EN460), an approximate one-log reduction in kon was observed, while koff was unchanged. This might indicate a more favorable ligand–analyte association interface for the VH–(GGGGS)3–VL than the VL–218–VH for this particular PEGylated scFv protein, although the overall conclusion is that each of the PEG–scFv compounds is functional. Binding kinetics for PEG–scFv C-terminal cysteine variants (e.g. EN454), in which the his6 tag does not precede a C-terminal cysteine, appear to produce faster kon and slower koff kinetics (data not shown).


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Table III. Binding analysis of the interaction of TNF-{alpha} ligand and scFv or PEG–scFv analyte compounds using surface plasmon resonance
 
Neutralization of TNF-{alpha} cellular cytotoxicity by scFv and PEG–scFv

We developed a selective and precise cell-based assay for quantitation of the neutralization of TNF-{alpha} by anti-TNF-{alpha} scFv that served as a functional screening analysis of the PEG–scFv compounds. Figure 3E shows results of cell rescue from TNF-{alpha} cellular cytotoxicity (the hallmark in vitro activity of this cytokine) by EN450 scFv and PEG–scFv. Table IV compiles the activities of representative compounds expressed as IC50 determinations. All scFv and PEG–scFv were effective in neutralization of TNF-{alpha} cytotoxic activity in a relatively narrow range of 16–191-fold molar excess of scFv over TNF-{alpha}. There was only a slight proclivity, in those PEG–scFv compounds having a his6-C attachment site, to display diminished neutralization activity compared with their unmodified scFv counterparts.


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Table IV. Neutralization of TNF-{alpha} cellular cytotoxicity by scFv and PEG–scFv compoundsa
 
Flow cytometry analysis of TNF-{alpha} cell receptor binding competition

Biotinylated huTNF-{alpha} was examined in WEHI-13VAR cell receptor binding assays by FACSCalibur analysis. Competition for receptor binding of TNF-{alpha} by each series of scFv and PEG–scFv (Table I) was evaluated by flow cytometry. Control compounds, biotinylated soybean trypsin inhibitor, CC49/218 scFv and PEG had no effect on TNF-{alpha} binding. As shown in Figure 4 for the EN450 compounds, PEG–scFv with 20 and 40 kDa PEG polymer mass completely eliminated TNF-{alpha} binding to cells at a molar ratio of 16:1 (PEG–scFv:TNF-{alpha}). At the same (or even 4-fold greater) molar concentration, unmodified EN450 scFv protein (and also EN450 5 kDa PEG–scFv) only partially reduced TNF-{alpha} binding to cells. A similar trend was observed for EN452, EN456 and EN460 compounds. Therefore, in these analyses, PEG–scFv proteins were more potent than native scFv proteins. A cross-examination of these preparations from the flow cytometric studies in the cell cytotoxicity rescue assays (wherein prolonged exposure to these compounds is provided) did not demonstrate a diminution in IC50 values; there is not, therefore, a selective inactivation of unmodified scFv proteins in the flow cytometry analysis conditions. Rather, it appears that, in these receptor-based analyses, the attached PEG polymer amplifies the neutralization potency of the TNF-{alpha}-binding scFv protein. We suggest that this may be a consequence of steric, diffusional or mechanistic hindrance of the efficiency of the release and transfer of a TNF-{alpha} trimer molecule, once bound by a PEG–scFv, on to its cell surface receptors. Alternatively, the slower koff rates recorded for some of the PEG–scFv compounds might play a role in this outcome. This hypothesis may be investigated in further experimentation, but considerable practical value may accrue if PEGylation of ligand-binding therapeutics can potentiate their neutralization capacity, since this is a major focus in modern biomedicine.



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Fig. 4. Flow cytometry analysis of biotinylated TNF-{alpha} binding to WEHI-13VAR cells in the presence of EN450 scFv or PEG–scFv. TNF-{alpha} was incubated with the scFv or PEG–scFv (16:1 molar ratio of scFv to TNF-{alpha}) prior to incubation with cells and streptavidin-PE for analysis on FACS Calibur. (A) scFv; (B) 20 kDa PEG–scFv; (C) 40 kDa PEG–scFv. Cell populations are shown without PE fluorescence labeling (1), with TNF-{alpha} and streptavidin-PE (2) or with TNF-{alpha} plus scFv or PEG–scFv plus streptavidin-PE (3).The shift towards low fluorescence intensity indicates reduced binding of TNF-{alpha} to the cells.

 
Antigenicity, stability and protease resistance

Antiserum raised in rabbits against either purified EN446 scFv protein or a synthetic 18-residue peptide with the 218 linker sequence provided reagents for immunoassays, including an evaluation of the antigenicity of the PEG–scFv conjugates. All PEG–scFv compounds demonstrated a marked reduction, but not elimination, of reactivity to anti-EN446 scFv serum. Anti-218 serum displayed minimal, but detectable, reactivity with the scFv proteins that were PEGylated on position 2 of the 218 linker. Possibly, an engineered central cysteine conjugation site in this linker would have totally eliminated this reactivity.

We did not find that the PEG–scFv conjugates demonstrated increased resistance to treatment with proteases in vitro. However, both scFv and PEG–scFv displayed prolonged plasma stability as assessed by IC50 bioactivity and western blotting and both scFv and PEG–scFv could be cycled through freeze–thaw or lyophilization with total recovery of bioactivity.

Pharmacokinetics of scFv and PEG–scFv in mice

Pharmacokinetic parameters of the EN450 and EN456 scFv and PEG–scFv conjugates were determined (Table V). ELISA protocols were developed for scFv quantitation as described in Materials and methods. Intravenous administration of EN450 40 kDa PEG–scFv resulted in a >200-fold increase in circulating life compared with the ethyl-scFv version (Figure 5A). Concomitantly, the AUC for EN450 40 kDa PEG–scFv exhibited an 800-fold increase compared with the ethyl-scFv. The Cmax of scFv and PEG–scFv did not show a correlation with the size of attached PEG polymer. Subcutaneous administration of EN450 20 kDa PEG–scFv demonstrated a 5.5-fold increased AUC when compared with intravenous administration of the same compound (Figure 5B). Although the Cmax decreased by 30% compared with i.v. administration, a nearly 6-fold increase in biological half-life was observed. The enormous prolongation of serum half-lives for PEG–scFv compounds addresses a limitation of scFv proteins as therapeutics due to their rapid renal elimination.


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Table V. Pharmacokinetic parameters of scFv and PEG–scFv compounds in micea
 


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Fig. 5. Pharmacokinetics of scFv and PEG–scFv in mice. (A) Intravenous administration at 4 mg scFv protein/kg for 40 kDa PEG–EN450 (open squares), 40 kDa PEG–EN456 (closed squares), 20 kDa PEG–EN450 (open circles), 20 kDa PEG–EN456 (closed circles), 5 kDa PEG–EN450 (asterisks), EN450 (open triangles), EN456 (closed triangles) and ethyl-EN450 (diamonds). Filled symbols represent EN456 compounds; open symbols represent EN450 compounds. (B) Subcutaneous administration at 4 mg scFv protein/kg for 20 kDa PEG–EN450 (open squares) and EN450 (closed squares); three mice per group.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Natural VL/VH antigen-binding domains – the Fv modules – of the human immunological repertoire are employed in a limited number of physical sizes and functional formats corresponding to the IgG, IgM, IgA, IgE and IgD macromolecules. A long-standing goal of antibody engineers is to extend the versatility of antigen-binding proteins by tailoring the binding properties, effector functions, valency and pharmacokinetic behavior of immunomolecules through protein and bioconjugate strategies. The single gene, single polypeptide VH/L–linker–VL/H design of single-chain Fv proteins provides a minimal antigen-binding format that has become widely utilized in phage-, ribosome- and yeast-display library technology. Although the isolation and affinity maturation of target-specific scFv proteins is now well established, the optimized scFv lead candidates are typically engineered into a full-length IgG protein prior to clinical development. However, the confinements of IgG Fc-derived effector functions, macromolecular configuration and programmed distribution do not match the preferred characteristics of every target neutralizing drug.

Six FDA licensed protein therapeutics and several compounds in ongoing clinical trials employ PEGylation to prolong circulating life and reduce immunogenicity (Harris and Chess, 2003Go). Although the concept of site-specific polymer conjugation has been established (Goodson and Katre, 1990Go; Wang et al., 1998Go; Chapman, 2002Go), the marketed PEG–protein drugs were developed with random PEG attachments on primary amines or selectively at the N-terminal amine. In our present study, we sought to apply the essential tenets of protein engineering research to the investigation of site-specific PEGylation of scFv proteins, through the design, production and rigorous structure–function analyses of selected anti-TNF-{alpha} PEG–scFv compounds.

In order to maximize surface exposure and avoid interactions with the antigen-binding site, placement of a single free cysteine was either at position 2 of the linkers (in either VL/VH or VH/VL orientations) or at the VH or VL C-terminus. In some of the variant proteins tested, we included an intervening six-histidine peptide between the authentic V domain C-terminus and the free sulfhydryl, in order not only to provide an affinity purification tag, but also to act as an extension segment that might further promote efficient conjugation to the bulky 20 and 40 kDa PEG–maleimide polymers (Figure 1). In practice, we found that conjugation efficiencies were comparable whether the free sulfhydryl was placed at the true VH domain C-terminus (EN454) or after the his6 tag (EN450). Since we found similar or more favorable binding kinetics and IC50 in the PEG–scFv constructions lacking the his6 tag, omission of this segment may be preferred in PEG–scFv development. Conjugation efficiencies and bioconjugate purification yields were also similar for the PEG–scFv variants with a linker sulfhydryl group (EN456, EN460). Peptide mapping, N-terminal protein sequencing, maleimide reactivity analysis and molecular mass determinations confirmed the highly selective attachment of PEG polymers to only the engineered thiol of the scFv variant proteins. One advantage of employing sulfhydryl directed moieties in scFv engineering, when compared with antibodies and proteins in general, is that free cysteines are uncommon in the Fv scaffold (Kabat et al., 1991Go) and the four disulfide bonded cysteines of the framework are completely buried (Padlan, 1994Go).

Unfortunately, however, reduced protein expression and increased aggregation in E.coli of designed scFv cysteine variants have been reported by several groups, e.g. Schmiedl et al. (Schmiedl et al., 2000Go). Our own investigations of E.coli secretion of these scFv cysteine variant proteins (Table I) resulted in moderate yields of both soluble and insoluble scFv in the periplasmic extracts. This encouraged us to develop an efficient process for recovery of soluble active scFv by secretion from P.pastoris. Using a designed minimal signal sequence from the yeast alpha mating factor with a conserved cleavage site, high-level secretion (50–100 mg/l) of each of the active scFv variant proteins was achieved. Interestingly, there was no trend toward reduced yields when a single free thiol was engineered into the scFv, but introduction of two free thiols did diminish secretion. We suggest that our production protocol for engineered proteins bearing free sulfhydryls may have applications beyond antibody engineering.

Bioactivities of this series of TNF-{alpha} binding PEG–scFv bioconjugates were investigated with surface plasmon resonance, immunoassays, flow cytometry and cellular cytotoxicity rescue. Although the overall outcome of these analyses is a demonstration of retained function in the PEGylated proteins, we observed some differing activity characteristics in the scFv and PEG–scFv compounds that may illuminate the properties of these structural formats. In BIAcore analysis, we found a trend toward slower association rates with increasing mass of attached polymer in PEG–scFv. For example, relative to the unmodified scFv, the 5 kDa EN450 and 40 kDa EN450 bioconjugates displayed about 4- and 40-fold slower on-rates, respectively. In contrast, the dissociation rates of these two compounds did not differ greatly from the unmodified scFv protein. Both the diminution of on-rates and retention (or even reduction) of off-rates were also perturbed by the scFv architecture, as seen in comparisons of variable domain orientation and/or choice of linker, as well as the PEG mass and attachment position. Physical hindrance by the bulky and mobile PEG constituent appears to retard the association with TNF-{alpha} bound to the CM5 chip, but does not substantially alter its dissociation. We would view both C-terminal and linker position 2 attachment sites as promising for future designs.

Establishment of a cell-based assay for neutralization of TNF-{alpha} cellular cytotoxicity, which is the most characteristic in vitro property of this cytokine, provided a precise method for both screening PEG–scFv compounds and for cross-analysis with other analytical methods. As we showed in Figure 3E and Table IV, comparisons of scFv proteins and their PEGylated counterparts indicated that both formats were similarly potent in cell rescue from TNF-{alpha} cytotoxicity. Next, we investigated the inhibition of cell receptor binding of biotinylated TNF-{alpha} by the PEG–scFv derivatives in flow cytometry measurements. All PEG–scFv compounds with 20 or 40 kDa polymers totally eliminated TNF-{alpha} binding to cells at concentrations that allowed only partial inhibition by either scFv or 5 kDa PEG–scFv. The flow cytometry data illustrate the capacity of PEGylated scFv to demonstrate enhanced potency, relative to native scFv, under defined assay conditions. Antigenicity analysis by immunoassays also illustrated the property of PEG–scFv proteins to demonstrate a markedly different response to plasma that was obtained from animals immunized with the scFv or linker segment.

We suggest that the observed functional alterations of potency for PEG–scFv compounds in the above assay methods may also have implications for the functional behavior of PEG–scFv therapeutics in vivo. The scFv neutralization or inhibition of ligand-receptor and other protein–protein target interactions might be meaningfully enhanced or diminished by the steric or mechanistic participation of the polymer constituent, while the local environment of the target might determine the degree of influence of the PEG polymer, if any. Since scFv proteins are also utilized in proteomic microarrays, our results may also have relevance to the design of polymer tethered diagnostics.

Insight into the correlation of structure to mechanism for the PEG–scFv compounds is provided by the SEC results (Table II). The hydrodynamic volumes of PEGylated proteins are expanded far beyond the increase in molecular mass due to the hydrophilicity and chain flexibility of this polymer. In SEC analysis, the 40 kDa PEG–scFv bioconjugates exhibit a predicted molecular weight nearly 10-fold greater than the molecular mass, as determined by MALDI-TOF-MS. This physical behavior of PEG–scFv in aqueous solution reveals a remarkable macromolecule and this magnification of hydrodynamic volume is also reflected in the pharmacokinetic analysis of the PEGylated scFv derivatives (Table V). PEGylated EN450 and EN456 scFv proteins demonstrated prolongation of biological half-lives that correlated with polymer molecular mass. The 40 kDa PEG–scFv derivatives exhibit circulating lives in the range of intact monoclonal antibodies. However, the benefits of PEGylation of scFv proteins might be fully realized by the capacity not merely to extend, but actually tailor, the pharmacokinetics of these antigen-binding proteins through polymer selection. In illustration of the capacity of PEG–scFv to provide a modular format for more complex designs, we have also been able to engineer the EN450 and EN456 scFv in a divalent format using bismaleimide–PEG compounds. In summary, our results indicate that novel scFv compounds may be designed through site-specific PEGylation and may provide an optional format to conventional therapeutic monoclonal antibodies.


    Acknowledgements
 
We thank Rich Greenwald and Steve Youngster for materials and advice. We also thank Pascal Bailon and Uli Grau for a critical review of the manuscript, and our colleagues at Micromet for encouragement and support.


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Received June 25, 2003; revised July 16, 2003; accepted July 20, 2003.





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