nature biotechnology • VOLUME 21 • MAY 2003 • www.nature.com/naturebiotechnology

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Site-specific PEGylation of a lysine-deficient TNF-α with full bioactivity

Yoko Yamamoto1*, Yasuo Tsutsumi1* , Yasuo Yoshioka1, Toshihide Nishibata1, Kyoko Kobayashi1, Takayuki Okamoto1,Yohei Mukai1, Tomoe Shimizu1, Shinsaku Nakagawa1, Satoshi Nagata2, and Tadanori Mayumi1

Published online 31 March 2003; doi:10.1038/nbt812

Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostlynonspecific and may occur at all lysine residues, some of which may be within or near an active site. ResultantPEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop astrategy for site-specific mono-PEGylation using tumor necrosis factor-α (TNF-α). We prepared phagelibraries expressing TNF-α mutants in which all the lysine residues were replaced with other amino acids. Afully bioactive lysine-deficient mutant TNF-α (mTNF-α-Lys(–)) was isolated by panning against TNF-α-neu-tralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-α-Lys(–)was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-α-Lys(–), with superiormolecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than ran-domly mono-PEGylated wild-type TNF-α. These results suggest the usefulness of the phage display systemfor creating functional mutant proteins and of our site-specific PEGylation approach.

Recent advances in structural genomics and pharmacoproteomicswill clarify the functions of numerous proteins. Therapeutic applica-tions of bioactive proteins, such as newly identified proteins andcytokines, are also promising1–3. Because these proteins are generallyquite unstable in vivo, their clinical application requires frequentadministration at high dosages. This often results in impaired home-ostasis in vivo and may cause severe adverse effects4. Cytokines suchas tumor necrosis factor-α (TNF-α) have diverse actions on varioustissues, making it difficult to obtain therapeutic effects with suchproteins while minimizing their side effects.

To overcome these problems, bioactive proteins have been conju-gated with water-soluble polymers such as PEG5,6. PEGylation ofproteins increases their molecular size and enhances steric hin-drance, both of which are dependent on PEG attached to the protein.These effects improve the plasma half-lives of proteins and their sta-bility against proteolytic cleavage, and also decrease their immuno-genicity. PEGylated interferon-α2a (IFN-α2a) and IFN-α2b havebeen used in clinical treatment of hepatitis C7,8. In addition, we havepreviously reported that PEGylation of proteins such as TNF-α9–11,interleukin-6 (IL-6)12,13, and immunotoxin14 could enhance thera-peutic potency and could reduce undesirable effects.

However, this approach is limited by the frequent substantial lossof protein specific activity associated with PEGylation15,16. Lysineamino groups of proteins are often used as substrates for PEGylationbecause they are highly reactive and the PEGylation reaction is mildenough to minimize disruption of the protein structure. ThisPEGylation, however, is nonspecific and occurs at the N terminus aswell as all internal lysine residues, some of which may be in or nearprotein active sites. Such PEGylated proteins are thus composed ofpositional isomers with PEG at various sites, which have distinct

activities and other characteristics. Such PEGylated isomer mixturescan therefore have inconsistent therapeutic effects17. To overcomethis problem, site-specific PEGylation can be achieved through a freethiol in the engineered proteins14,18. The efficacy of this approach iscompromised, however, by an extremely low yield of PEGylated pro-teins and a substantial loss of activity owing to the introduction of afree thiol residue19.

Here we have attempted to develop a strategy for site-specificPEGylation to overcome these drawbacks. Our model protein wasTNF-α, a promising new drug for cancer therapy with toxic sideeffects that limit its clinical application as a systemic antitumoragent20–22. Because a deletion mutant of TNF-α lacking eightresidues at the N terminus retains full bioactivity, we surmised thatthe N terminus of TNF-α is not important for function and mighttherefore be a good target for PEGylation23. However, amino groupsof all six internal lysine residues in TNF-α are also targets forPEGylation, and studies using site-directed mutagenesis analysishave reported that Lys11 and Lys90 are vital for its bioactivity24,25.Nevertheless, if one could construct fully bioactive TNF-α in whichall the lysine residues were replaced with other amino acids, site-specific PEGylation of the N terminus could then be carried out.This PEGylated mutant TNF-α should have excellent molecularuniformity and retain high bioactivity.

Phage libraries expressing polypeptides, such as naive antibodiesor random peptides, have extensively been applied to the identifica-tion of specific molecules with high affinity for a targeted lig-and26–28. In this study, we first applied the phage display system tocreate fully bioactive lysine-deficient mutant TNF-α, and then car-ried out site-specific PEGylation for improving therapeutic potency.This N-terminal mono-PEGylated mutant TNF-α had comparable

1Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. 2Laboratory ofMolecular Biology, Division of Basic Science, National Cancer Institute, National Institutes of Health, Building 37, Room 4E16, 37 Convent Drive MSC 4255,

Bethesda, MD 20892-4255, USA. *These authors contributed equally to the work. †Corresponding author (tsutsumi@phs.osaka-u.ac.jp).

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www.nature.com/naturebiotechnology • MAY 2003 • VOLUME 21 • nature biotechnology 547

bioactivity to unmodified wild-type TNF-α in vitro, and other prop-erties including plasma half-life, antitumor activity, and toxicitywere greatly improved.

ResultsConstruction of a phage library displaying mutant TNF-α (mTNF-α). To create fully bioactive lysine-deficient mTNF-α with substitu-tion of all six lysine residues, we prepared a cDNA library of mTNF-α with randomized sequences in place of the six lysine codons.Three-step PCR was carried out to replace the lysine codons ran-domly with an ‘NNS’ sequence (Fig. 1). Construction of the phagelibrary expressing mTNF-α fused with the phage g3p is described inthe Experimental Protocol. This phage library consisted of 5 × 107

independent recombinant clones. DNA sequencing analysis of 12randomly picked clones confirmed the predicted diversity of thelibrary (Table 1A). Among these 12 phage clones, we found clonesencoding mTNF-α without bioactivity or without ability to formtrimeric structure.

Library selection. To isolate lysine-deficient mTNF-α that retainsits full bioactivity, the phage library was subjected to two rounds ofpanning against neutralizing anti-TNF antibody or TNF receptor-I(TNF-RI) using a BIAcore biosensor. We randomly picked 480 phageclones after the second panning, and screened the clones by ELISAusing immobilized neutralizing anti-TNF-α antibody, which recog-nizes the binding site of TNF-α to TNF-RI, and by cytotoxicity assayusing LM cells for its TNF-specific bioactivity. Among the 480

clones, 197 had reactivity similar to that of wild-type TNF-α. DNAsequencing analysis of these positive clones showed that 6 of theclones, including the lysine-deficient mTNF-α 2-11 (Table 1B),lacked all six lysine residues. The replaced amino acids in these sixclones were diverse. All six had bioactivity, presumably throughbinding to TNF-RI, when analyzed by cytotoxicity assay using mouseLM cells, a cell line derived from L929 cells.

Among the six clones, the fully bioactive mTNF-α 2-11 clone(mTNF-α-Lys(–)) was chosen for further analyses, because therecombinant mTNF-α-Lys(–) could be most efficiently produced bya general recombinant protein technology. Analysis of the proteinsby gel filtration chromatography and ultracentrifugation indicatedthat purified mTNF-α-Lys(–) could form a trimeric structure similarto those of wild-type TNF-α and natural human TNF-α in aqueoussolution (data not shown). mTNF-α-Lys(–) had a specific activityequal to that of wild-type TNF-α or commercially available recom-binant TNF-α (Fig. 2A). To assess further the specific bioactivitythrough TNF-RI, we conducted a cytotoxicity assay using mouseL929 cells in the presence of actinomycin D at the concentration of2 µg/ml. We found that the LC50 values (that is, the concentrationscapable of killing 50% of cells) of wild-type TNF-α and mTNF-α-Lys(–) were 0.51 ± 0.05 ng/ml and 0.48 ± 0.07 ng/ml, respectively.When growth stimulation of MOLT-4, a lymphoma cell derivedfrom human T cells, was analyzed, mTNF-α-Lys(–) had activity sim-ilar to that of wild-type TNF-α (ED50 (half-maximal effective dose)of wild-type TNF-α = 13.80 ± 0.44 ng/ml, ED50 of mTNF-α-Lys(–) =12.59 ± 0.52 ng/ml). Therefore, it appears that mTNF-α-Lys(–) hasfull bioactivity through the binding to TNF receptor-II (TNF-RII).The affinity of mTNF-α-Lys(–) for TNF-RI or for TNF-RII was alsomeasured using BIAcore, and compared to that of wild-type TNF-α.The Kd between mTNF-α-Lys(–) and TNF-RI or TNF-RII was 1.72 ±0.45 × 10–9 M or 7.48 ± 0.39 × 10–10 M, respectively, and the Kd

between wild-type TNF-α and TNF-RI or TNF-RII was 1.67 ± 0.25 ×10–9 M, or 7.23 ± 0.32 × 10–10 M, respectively. The number of prima-

Table 1A. Nucleotide and amino acid sequences of mTNF-αforms encoded by phage clones randomly isolated from thephage library

Clone PositionK11 K65 K90 K98 K112 K128

A E (GAG) L (TTG) T (ACC) N (AAC) R (AGG) V (CTC)B V (GTG) C (TGC) A (GCC) P (CCG) G (GGC) H (CAG)C W (TGG) F (TTC) T (ACG) R (AGG) L (CTC) S (AGC)D R (CGG) C (TGC) K (AAG) Y (TAC) P (GAC) V (GTC)E E (GAG) V (GTC) A (GCG) T (ACG) P (CCG) P (CCC)F W (TGG) F (TTC) H (CAC) T (ACG) T (ACC) W (TGG)G V (GTC) G (GGG) K (AAG) S (TCG) T (ACC) G (GGC)H M (ATG) G (GGG) H (CAG) K (AAG) R (AGG) K (AAG)I C (TGC) L (TTG) P (GAC) R (CGG) R (CGC) H (CAC)J I (ATC) P (CCG) V (GTC) P (GAC) Q (CAG) P (CCC)K L (TTG) L (CTC) D (GAC) P (CCC) S (TCG) Q (CAG)L L (CTC) L (TTG) T (ACC) T (ACG) S (TCC) Q (CAG)

Table 1B. Nucleotide and amino acid sequences of lysine-defi-cient mTNF-α forms obtained after two rounds of affinity pan-ning

Clone PositionK11 K65 K90 K98 K112 K128

2-11 M (ATG) S (TCG) P (CCC) R (CGC) N (AAC) P (CCG)2-29 L L T T S Q2-64 L R A A A P2-87 A T G R L L2-144 I L R D N Q2-153 A S P S T E

Figure 1. Construction of a phage library that displays lysine-deficientmTNF-α in which all lysine residues are replaced with other amino acids.Lysine codons were replaced with the randomized sequence ‘NNS’.Mutations were introduced at the six lysine codons in TNF-α by three-stepPCR using the plasmid pY02-TNF, encoding human TNF-α, as templateand four oligonucleotides (oligos 1–4) encoding NNS (where N and Srepresent G/A/T/C or G/C, respectively, at each lysine codon). NNSencodes all 20 different amino acids. After the third PCR, products inwhich all six lysine codons had been replaced with NNS were ligated withthe phagemid vector pY02 for the display of mTNF-α forms on the phagesurface as fusion proteins with g3p.

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ry amine groups in mTNF-α-Lys(–), as determined by a fluo-rescamine assay, decreased to about one-seventh of that in wild-typeTNF-α (Fig. 2B), confirming the absence of lysine residues inmTNF-α-Lys(–).

Site-specific PEGylation of mTNF-α-Lys(–). Wild-type TNF-αand mTNF-α-Lys(–) were modified with activated PEG5K.PEGylated TNF-α molecules were detected by SDS-PAGE using acombination of Coomassie blue staining for protein and bariumiodide staining for PEG. In experiments shown in lanes 4 and 5(Fig. 3A), wild-type TNF-α molecules were treated with 5-fold and50-fold molar excess of activated PEG against total primary aminegroups of wild-type TNF-α, respectively. The PEGylation conditionof wild-type TNF-α shown in lane 5 is the same as that of mTNF-α-Lys(–) shown in lane 3. In both of these experiments, a 50-fold molarexcess of activated PEG was used. This result suggested that a singlePEG molecule was attached selectively to the N terminus of mTNF-α-Lys(–), whereas PEG molecules were introduced randomly at mul-tiple positions in the wild-type TNF-α. TNF-α molecules conjugatedwith one PEG molecule were purified by gel filtration. Purified site-specific mono-PEGylated mTNF-α-Lys(–) (sp-PEG-mTNF-α) andrandomly mono-PEGylated wild-type TNF-α (ran-PEG-TNF-α)were examined for their specific bioactivity (Fig. 3B). sp-PEG-TNF-αhad ∼ 80% of the specific activity of mTNF-α-Lys(–), whereas ran-PEG-TNF-α had <10% of the specific activity of wild-type TNF-α.

Toxicity and antitumor activity. To assess toxicity, the TNF-αmolecules were intravenously injected at various doses into mice.The LD50 values for wild-type TNF-α, mTNF-α-Lys(–), ran-PEG-TNF-α, and sp-PEG-mTNF-α were 389, 427, 1,287, and 1,404 µgprotein/kg, respectively. The antitumor activity of the TNF-α mole-cules against Meth-A solid tumors was also assessed by single intra-venous (i.v.) injection (Fig. 4), and the tumor hemorrhagic necroticarea was measured 24 hours after the injection (Fig. 4A). In micetreated with wild-type TNF-α, hemorrhagic necrosis was caused at10 mg/mouse. However, all mice treated with wild-type TNF-α at thisdose died within 24 hours after the injection. In mice treated withran-PEG-TNF-α, hemorrhagic necrosis was induced at 3 mg/mouse,and sudden death was not observed at 10 mg/mouse. But completetumor regression, defined as disappearance of tumor withoutregrowth within 90 days, was not observed. In mice treated withmTNF-α-Lys(–) at a dose of 10 mg/mouse, an area was observed thatwas ∼ 40% necrotic. Thus, antitumor activity of mTNF-α-Lys(–) washigher than that of wild-type TNF-α, although the in vitro specificactivity of the two molecules was the same. However, all the micetreated with mTNF-α-Lys(–) at a dose of 10 mg/mouse died within24 hours after the injection, and complete tumor regression was notobserved. In contrast, induction of tumor necrosis by sp-PEG-mTNF-α at a dose of 1 mg/mouse was as effective as that by wild-type TNF-α at 10 mg/mouse. Tumor growth was markedly inhibitedby sp-PEG-mTNF-α (Fig. 4B), and complete tumor regression wasobserved in 70% of mice treated at a dose of 3 mg/mouse.

Stability and pharmacokinetics of mono-PEGylated TNF-α mol-ecules. The stability of the TNF-α molecules was examined by incu-bating them with mouse serum at 37 °C for various periods of time(Fig. 5A). Although the bioactivity of both forms of unmodifiedTNF-α rapidly diminished, both mono-PEGylated forms of TNF-αwere more stable. The plasma concentration was also measured afterthe i.v. injection of a single dose of 1 mg each into mice (Fig. 5B). Theplasma half-life of mTNF-α-Lys(–) was 6 minutes, longer than thatof wild-type TNF-α (4 minutes), and the area under the curve(AUC) of mTNF-α-Lys(–) was twofold higher than that of wild-typeTNF-α. Plasma half-lives of both the mono-PEGylated forms ofTNF-α were longer than those of the unmodified TNF-α forms. Theplasma half-life of sp-PEG-mTNF-α was 40 minutes and was abouttenfold higher than that of wild-type TNF-α.

DiscussionOne of the most efficient ways of improving therapeutic potency ofproteins has been to modify them with PEG. PEGylated IFN-α hasclinically been shown to have marked antiviral activity againsthepatitis C7,8. However, clinical application of most PEGylated pro-teins has not yet been successful. In most cases, PEGylation occursrandomly at multiple lysine residues in proteins, some of whichmay be located in or near the protein active site15. The resultantPEGylated proteins are therefore heterogeneous and composed ofvarious positional isomers with distinct specific activities17.Additionally, their bioactivity is markedly lower than that of theunmodified proteins. To overcome these drawbacks, we have heredeveloped a PEGylation system in which the N terminus of the pro-tein is specifically PEGylated after creation of lysine-deficientmutants with full bioactivity through the use of phage libraries.

Phage display is a useful technique for constructing librariesexpressing millions of different peptides or proteins. In many cases,these phage libraries have been used in identifying ligands for pep-tide receptors, epitopes for monoclonal antibodies, and specificantibody repertoires26–28. We first applied the technique to screenfully bioactive lysine-deficient mTNF-α molecules that had all the

Figure 2. Characterization of isolated forms of lysine-deficient mTNF-α.(A) In vitro bioactivity of wild-type (WT) TNF-α and mTNF-α-Lys(–). Thespecific activity of TNF-α forms was measured by cytotoxicity assay usingLM cells in the presence of actinomycin D. Each data value represents themean ± s.d. IC50 is the concentration of wild-type TNF-α or mTNF-α-Lys(–) required for 50% inhibition of cell viability. Purchased recombinanthuman TNF-α was used as a standard. (B) Numbers of primary aminegroups in wild-type TNF-α and mTNF-α-Lys(–) as quantified by afluorescamine assay. Each value represents mean ± s.d.

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lysine residues replaced with other amino acids (Fig. 2). By affinityselection of clones with neutralizing anti-TNF-α antibody andwith TNF-RI, we successfully isolated lysine-deficient mutants withfull bioactivity. Site-directed mutagenesis analysis of TNF-α24,25

suggested that among the six lysine residues, Lys11 and Lys90 wereessential for the protein’s homotrimer formation as an active formand for interaction with its receptor, respectively. Therefore, we arecurrently investigating how mTNF-α-Lys(–) completely retains itsbioactivity after the substitution of all of the lysine residues. It isgenerally thought that the replacement of lysine residues withamino acids other than basic ones such as arginine is not possiblewithout loss of bioactivity, because lysine residues have criticalfunctions in protein structure or ligand-receptor binding. Indeed,this is supported by the present results, in which some of the lysine-deficient mTNF-α molecules tested (Table 1A) completely lostbioactivity. In contrast, mTNF-α-Lys(–) fully retained its originalactivity after its lysine residues were replaced with methionine,proline, and other residues. To address this question, we have start-ed three-dimensional structural analysis of the protein and molec-ular analysis of the ligand-receptor interaction. Analysis usingBIAcore and using ultracentrifugation suggests that mTNF-α-Lys(–) has an affinity for TNF-RI and TNF-RII equivalent to that ofwild-type TNF-α. It also appears, from analysis using gel filtration

chromatography and ultracentrifuga-tion, that mTNF-α-Lys(–) has the sametrimeric structure as the wild-type pro-tein. Computer analysis of the three-dimensional structure of wild-typeTNF-α, in which all of the lysineresidues were replaced with the sameresidues as those observed in mTNF-α-Lys(–) suggested that the lysinereplacement did not induce a substan-tial structural change (data not shown).These results lead us to believe that wehave successfully isolated mTNF-α-Lys(–) with full bioactivity and correcttertiary structure, through the affinityselection using TNF-RI and TNF-α-neutralizing antibody, from the phagelibrary consisting of 5 × 107 variantTNF-α with replacement of all the sixlysine residues with others. We thinkthat the most important points for thesuccessful isolation of mTNF-α-Lys(–)are the construction of the library con-sisting of a large number of variantclones as well as the affinity panning. Wehave also isolated clones that retain asingle lysine residue and have greaterspecific activity and in vivo antitumoractivity than wild-type TNF-α. Thisresult may suggest that various func-tional mutants of TNF-α, such assuperagonists and antagonists of TNFreceptors, may be isolated by using thephage display system.

mTNF-α-Lys(–) was site-specificallyconjugated at its N terminus with onePEG5K molecule (Fig. 3). The maximalyield of sp-PEG-mTNF-α was >90%.ran-PEG-TNF-α was heterogeneous atthe molecular level and had <10% ofthe specific bioactivity of unmodified

wild-type TNF-α. By contrast, sp-PEG-mTNF-α was uniform at themolecular level and had similar bioactivity (80%) to that of unmod-ified mTNF-α-Lys(–). Additionally, from repeat chromatographicanalysis and cytotoxicity assay using LM cells, we found that sp-PEG-mTNF-α in PBS was stable for two months at –80 °C, for twoweeks at 4 °C and for 24 hours at room temperature. When we used a40 kDa branched PEG, mono-PEGylated mTNF-α-Lys(–) had 70%the bioactivity of the unmodified form (data not shown). Theseresults indicate the usefulness of our PEGylation system. RandomlyPEGylated IFN-α2b, IFN-α conjugated with 40 kDa of branchedPEG, is known from clinical use to have ∼ 10% of its bioactivity ascompared to unmodified IFN-α. Because the N terminus of IFN-αis not important for its function, we are now applying ourPEGylation system to the modification of IFN-α.

sp-PEG-mTNF-α had approximately ten times higher antitu-mor potency than wild-type TNF-α against the murine Meth-Afibrosarcoma model (Fig. 4). Furthermore, toxicity of sp-PEG-mTNF-α was similar to that of ran-PEG-TNF-α , and was ∼ 3-foldlower than those of unmodified wild-type TNF-α and mTNF-α-Lys(–). Because the antitumor activity of sp-PEG-mTNF-α was10-fold and 3-fold higher than those of wild-type TNF-α and ran-PEG-TNF-α , respectively, its therapeutic window may be greatlyimproved. However, the replacement of the lysine residues in sp-

Figure 3. Characterization of site-specific PEGylated-mTNF-α-Lys(–). (A) SDS-PAGE analysis ofPEGylated forms of TNF-α. Wild-type TNF-α or mTNF-α-Lys(–) was mixed with 5-fold (lane 4) or 50-fold (lanes 3 and 5) molar excess of activated PEG5K against total primary amine groups of TNF-αforms at 37 °C for 30 min. SDS-PAGE analysis of TNF-α and PEGylated TNF-α forms was conductedunder reducing conditions. The two SDS gels were run under identical conditions, and proteins andPEG were stained using Coomassie blue (top) and barium iodide (bottom), respectively. Lane M,prestained molecular weight standards; lane 1, mTNF-α-Lys(–); lane 2, wild-type TNF-α; lane 3, site-specifically PEGylated mTNF-α-Lys(–); lane 4, randomly PEGylated wild-type TNF-α (5-fold molarexcess of PEG); lane 5, randomly PEGylated wild-type TNF-α (50-fold molar excess of PEG); lane 6,PEG5K. (B) In vitro bioactivity of mono-PEGylated forms of TNF-α. Each data value represents themean ± s.d.

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PEG-mTNF-α may increase the immunogenicity of the protein,which must be investigated, though on the other hand, it is knownthat PEGylation considerably decreases proteins’ immunogenici-ty14. Thus, sp-PEG-mTNF-α may have clinical utility in cancertherapy.

It is interesting to consider why the two forms of mono-PEGylated TNF-α (sp-PEG-mTNF-α and ran-PEG-TNF-α) havehigher antitumor activity and lower toxicity. In general, the perme-ability of tumor vasculature is enhanced as compared with that ofnormal tissues, so macromolecules such as albumin may accumu-late in the tumor. In addition, it is known that TNF-α selectivelyenhances the vascular permeability of macromolecules in tumortissues29–31. However, 3 hours after i.v. injection into Meth-Afibrosarcoma tumor-bearing mice, we found that there was no sub-stantial difference in tumor distribution between both mono-PEGylated and unmodified forms of TNF-α (data not shown). Theantitumor effect of TNF-α is known to result not only from itsdirect cytotoxicity against tumor cells but also from activation ofantitumor effector immune cells in the blood, such as macrophagesand cytotoxic lymphocytes. Furthermore, TNF-α may cause specif-ic damage to tumor blood vessels11. Therefore, we postulate that theenhanced antitumor activity of mono-PEGylated forms of TNF-α(especially sp-PEG-mTNF-α) may be due to the tenfold enhanced

retention in blood as compared with unmodified forms of TNF-α(Fig. 5B). By contrast, the distribution of TNF-α from blood intothe adversely affected tissue, such as the liver, causes toxic sideeffects. The improved retention of TNF-α in the vascular space andthe resultant decrease in transfer of TNF-α to these tissues isexpected to reduce the side effects. In fact, the distribution of bothforms of mono-PEGylated TNF-α to liver was only ∼ 70% that ofunmodified forms of TNF-α, probably because of the larger molec-ular sizes of the PEGylated forms. Although the detailed mecha-nism of the side-effect reduction resulting from PEGylation is notclear, reduced tissue distribution of both forms of mono-PEGylated TNF-α seemed to diminish the side effects somewhat.

In conclusion, phage display is a useful tool to create functionalmutants, as shown by the production of a fully bioactive mTNF-αlacking all lysine residues. Site-specific PEGylation to the N termi-nus of proteins may be able to overcome the problems ofPEGylation, such as lower bioactivity, lower yield, and poor molec-ular uniformity, as well as increase therapeutic potency. We are cur-rently trying to construct lysine-deficient forms of IFN-α, TNF-RI,antibody specific to TNF-α (scFv), and IL-6 with full activity andsite-specific PEGylation of their N termini. After constructinglysine-deficient molecules, their site-specific PEGylation may per-mit the production of drugs with higher efficacy.

Figure 5. Stability of various forms of PEGylated TNF-α. (A) Stability ofmono-PEGylated forms of TNF-α in mouse serum. The stability of TNF-αor mono-PEGylated TNF-α forms was determined by incubating at 37 °Cin 75% mouse serum from female BALB/c mice. The amount of activeTNF-α remaining after different periods of incubation times wasdetermined by a cytotoxicity assay using LM cells. (B) Pharmacokineticsof TNF-α and mono-PEGylated TNF-α forms after their i.v. injection. Theconcentration of TNF-α molecules in serum was quantified by ELISA. Astandard curve was made for each form of TNF-α. Each data valuerepresents the mean ± s.e.m. WT, wild type.

Figure 4. Antitumor effects of various forms of TNF-α on mice bearingintradermally implanted Meth-A solid tumors. (A) Tumor necrotic effects ofi.v.-administered TNF-α and mono-PEGylated TNF-α forms.Tumorhemorrhagic necrosis was scored 24 h after the injection. Each data value isthe mean ± s.e.m. N.D., not detected. Sudden death was defined as deaththat occurred within 24 h after the i.v. injection. (B) Antitumor effect of TNF-αor mono-PEGylated TNF-α forms on Meth-A solid tumors. At 7 d after thetumor inoculation, TNF-α or mono-PEGylated TNF-α forms were given by i.v.injection. Data are expressed as relative tumor volumes at a given time/meantumor volume on day 7. Each data value represents the mean ± s.d.

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Experimental protocolMaterials. Methoxy-PEG-succinimidyl propionate (mPEG-SPA; Mr 5000)was purchased from Shearwater Polymers (Huntsville, AL). Other reagentsand solvents were obtained from standard sources.

Library construction. Plasmid pY02-TNF encoding human TNF-α, in whichthe C terminus of TNF-α was fused to the N terminus of M13 phage g3p, wasused as a template for PCR. Oligos 1–4 were designed to have the sequence‘NNS’, where N and S represent G/A/T/C and G/C, respectively, at TNF-αcodons for Lys11, Lys65, Lys90, Lys98, Lys112, and Lys128.

Oligo 1: 5′-TCTACTCCCAGGTCCTCTTCNNSGGCCAAGGCTGCCC-CTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCATCGC-CGTCTCCTACCAG-3′

Oligo 2: 5′-GGCCTCAGCCCCCTCTGGGGTCTCCCTCTGGCAGGGG-CTSNNGATGGCAGAGAGGAGGTTGACSNNGGTCTGGTAGGAGACG-GCGATGCG-3′

Oligo 3: 5′-TAGTCGGGCCGATTGATCTCAGCGCTGAGTCGGTCACC-SNNCTCCAGCTGGAAGACCCCTCCCAGATAGATGGGCTCAT-ACCAGGGSNNGGCCTCAGCCCCCTCTGGGGT-3′

Oligo 4: 5′-TAGTTGTTCCTTTCTATGCGGCCCAGCCGGCCATGGC-CATGGTCAGATCATCTTCTCGAACCCCGAGTGACNNSCCTGTAGCC-CATGTTGTAGCA-3′

The sequence NNS can encode all 20 amino acids. The mutagenic oligonu-cleotides were used for three-step PCR amplification of the TNF-α cDNA.The products of the third PCR were digested with restriction enzymes andligated with the phagemid vector pY02 so as to display mTNF-α molecules onthe phage surface as fusion proteins with g3p. The phage library displayingmTNF-α molecules was prepared as described31.

Selection of phages displaying lysine-deficient mTNF-α with full bioactivi-ty. Neutralizing anti-TNF-α antibody or soluble human TNF-RI (110 µleach, 50 mg/ml in 10 mM sodium acetate buffer, pH 4.0) was immobilized toa sensor chip F1 surface using an amine coupling kit (BIAcore, Uppsala,Sweden), which resulted in an increase of 1,000–3,000 refractive index units(RIU). The phage library (50 µl) was injected at 3 µl/min over the sensor chip.After binding until the association phase was reached, the sensor chip waswashed with HBS buffer (10 mM HEPES, 3.4 mM EDTA, and 150 mM NaCl,pH 7.4) containing 0.05% Tween-20 and 0.1% BSA. Regeneration was carriedout using 15 µl 10 mM glycine-HCl. Eluate was collected and neutralizedwith Tris-HCl. The eluate phages were amplified and used for second-roundselection. Each phage selectively bound to anti-TNF-α antibody or TNF-RIwas isolated. The affinity of each mTNF-α encoded by the phage clone forneutralizing anti-TNF-α antibody and its bioactivity was examined byphage-ELISA and cytotoxicity assay using LM cells9, respectively.

Expression and purification of TNF-α forms. Plasmids pYas1-TNF andpYas1-mTNF encoding the human TNF-α and mTNF-α-Lys(–) gene, respec-tively, under the control of a T7 promoter, were prepared. Wild-type TNF-αand mTNF-α-Lys(–) proteins were produced in Escherichia coli BL21(DE3)harboring the expression plasmid pYas1-TNF or pYas1-mTNF. Both TNF-αproteins accumulated in the cells as an inclusion body. These inclusion bodiesprepared from cell paste by lysis were washed in nonionic detergent (Triton X-100) and solubilized in 6 M guanidine-HCl, 0.1 M Tris-HCl, pH 8.0, and 2 mMEDTA. Solubilized protein at 10 mg/ml was reduced with 10 mg/ml dithioery-thritol for 4 h at room temperature and refolded by 100-fold dilution in arefolding buffer containing 100 mM Tris-HCl, 2 mM EDTA, 0.5 M arginine,

and oxidized glutathione (551 mg/L). The refolding buffer was adjusted to pH10.5. The TNF-α proteins were allowed to refold for 36 h at 10 °C. After dialy-sis with 20 mM Tris-HCl, pH 7.4, containing 100 mM urea, active trimericproteins were purified from the refolding solution by ion-exchange column(Q-Sepharose and MonoQ) chromatography and size-exclusion chromatog-raphy (Superose 12, Amersham Pharmacia, Piscataway, NJ). Protein concen-trations were determined by using Coomassie Plus Protein Assay Reagent(Pierce, Rockford, IL). Endotoxin levels ware determined to be <300 pg/mgeach in the wild-type TNF-α and mTNF-α-Lys(–) preparations.

PEGylation of TNF-α. Wild-type TNF-α and mTNF-α-Lys(–) in PBS werereacted with 5-fold (wild-type TNF-α) or 50-fold (both TNF-α forms) molarexcess of mPEG5K-SPA against total primary amine groups of wild-typeTNF-α at 37 °C for 30 min. Then ε-aminocaproic acid (10-fold molar excessagainst the mPEG5K-SPA) was added to stop the reaction. SDS-PAGE analy-sis of PEGylated TNF-α forms was conducted under reducing conditions,and the gels were stained specifically for proteins or PEG32. ran-PEG-TNF-αand sp-PEG-mTNF-α were purified by size-exclusion chromatography(Superose 12, Amersham Pharmacia) in PBS. The specific bioactivities andaffinity for TNF-RI of mono-PEGylated forms of TNF-α were examined byLM cytotoxicity assay and BIAcore analysis, respectively. To quantify primaryamine groups, purified TNF-α and mTNF-α-Lys(–) were diluted with boratebuffer and then admixed with fluorescamine solution. Fluorescence intensity(390 nm excitation, 475 nm emission) was measured.

Antitumor studies. All experimental protocols for animal studies were inaccordance with the Principles of Laboratory Animal Care33. The antitumoreffects of various TNF-α forms were evaluated in mice bearing Meth-Afibrosarcoma. Meth-A cells were implanted intradermally (2 × 105 cells/site)in 5-week-old female BALB/c mice (n = 7). On day 7, when the tumor diam-eter reached 7 mm, TNF-α molecules were administered by a single i.v. injec-tion. Antitumor potency was estimated from the tumor volume and tumorhemorrhagic necrosis within 24 h after the injection. The tumor volume wascalculated using a formula described by Haranaka et al34.

Stability and pharmacokinetic assays. The stability of various forms of TNF-α was determined by incubating with 75% mouse serum at 37 °C. After differ-ent incubation periods, the remaining specific activity was determined by acytotoxicity assay against LM cells. For pharmacokinetic assay, normal femaleBALB/c mice were injected i.v. with 1 mg each of various forms of TNF-α.Blood samples were drawn at different times after the injection. The concen-trations of TNF-α forms in the blood samples were measured by ELISA.

AcknowledgmentsThis study was supported in part by a Grant-in-Aid for Scientific Research (No.13672385) from the Ministry of Education, Science and Culture of Japan, andin part by Health Sciences Research Grants for Research on Health Sciencesfocusing on Drug Innovation from the Japan Health Sciences Foundation(KH63124). We thank Ira Pastan (US National Cancer Institute, NationalInstitutes of Health) for advice and discussion, and Kenneth Santora (a memberof Ira Pastan’s lab at NCI/NIH) for editorial assistance.

Competing interests statementThe authors declare that they have no competing financial interests.

Received 14 August 2002; accepted 2 January 2003

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RESEARCH ARTICLE

nature biotechnology • VOLUME 21 • MAY 2003 • www.nature.com/naturebiotechnology552

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