Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional conjugation as targeting ligands

Miriam Bach1, Peter Hölig1, Eva Schlosser1, Tina Völkel1, Andreas Graser1, Rolf Müller2 and Roland E. Kontermann1,3

1vectron therapeutics AG, Rudolf-Breitscheid-Straße 24, 35037 Marburg and 2Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Emil-Mannkopff-Straße 2, 35033 Marburg, Germany

3 To whom correspondence should be addressed. e-mail: kontermann{at}vectron-ag.com


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ligand-targeted anticancer therapeutics represent an opportunity for the selective and efficient delivery of drugs to tumours. The chemical coupling of ligands to drugs or drug carrier systems is, however, often hampered by the presence of multiple reactive groups within the ligand, for example, {epsilon}-NH2 groups in lysine side chains. In this paper, we describe the isolation by phage display of human epidermal growth factor (EGF) variants without lysine and a reduced number of arginine residues. The selection on A431 carcinoma cells also revealed that R41 is indispensable for EGF binding activity as all EGF variants contained an arginine residue at this position. One EGF variant (EGFm1) with K28Q, R45S, K48S and R53S mutations was expressed in bacteria and showed an identical binding activity as wild-type EGF. EGFm1 could be labelled with fluorescein isothiocyanate demonstrating the accessibility of the N-terminal amino group for coupling reagents. Furthermore, coupling of EGFm1 to PEGylated liposomes resulted in target cell-specific binding and internalization of the liposomes. These human EGF variants should be advantageous for the generation of anticancer therapeutics targeting the EGF receptor, which is overexpressed by a wide variety of different tumours.

Keywords: EGF receptor/epidermal growth factor/liposomes/mutagenesis/phage display/targeting


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The concept of actively targeting drugs to tumour cells has led to the development of highly selective and efficacious anticancer therapeutics with reduced side effects (Allen, 2002Go). Tumour cell specificity is conferred by ligands conjugated to the drug or drug carrier system. A wide variety of ligands have been explored including natural ligands, antibodies, peptides, and carbohydrates (Forssen and Willis, 1998Go; Allen, 2002Go). Recent studies have shown that binding and subsequent internalization of actively targeted therapeutics by the target cells is a prerequisite to induce specific and efficacious antitumour effects (Park et al., 2002Go). Natural ligands such as growth factors and cytokines recognize cell surface-displayed receptors leading to receptor activation and internalization. Thus, these molecules are ideally suited for the generation of targeted internalizing drug systems.

Epidermal growth factor (EGF) is a monomeric protein consisting of 53 residues which binds specifically and with high affinity to the EGF receptor (Carpenter and Cohen, 1990Go; Lemmon et al., 1997Go). The EGF receptor is an attractive target for tumour therapy as it is overexpressed by a wide variety of human carcinomas, including cancers of the lung, liver, breast, head, neck, ovary and bladder (Salomon et al., 1995Go; Baselga, 2001Go; Ciardiello and Tortora, 2001Go; Nicholson et al., 2001Go). Various conjugates consisting of EGF and cytotoxic drugs (e.g. doxorubicin, carminomycin, phthalocyanine), ribonuclease, Pseudomonas exotoxin A, or radionuclides already demonstrated increased and selective delivery of the drugs into EGFR-expressing cells and the potential to inhibit tumour growth in animal models (Lee et al., 1993Go; Jinno et al., 1996Go; Lutsenko et al., 1999Go, 2000Go, 2002Go; Chen et al., 2002Go). Furthermore, liposomes and viral vectors displaying EGF on their surface were developed for target cell-specific drug or gene delivery (Kikuchi et al., 1996Go; Kullberg et al., 2002Go, 2003Go). In addition, targeting of drugs or toxins to EGF receptor-expressing tumour cells was also described for anti-EGFR antibody fusion proteins or conjugates (Aboud-Pirak et al., 1989Go; Schmidt et al., 1997Go).

Ligands are routinely conjugated to drugs or carrier systems by chemical coupling. However, the presence of several reactive groups at the ligand surface can lead to aggregation due to multiple cross-linking between ligands and/or carriers or to ligand inactivation due to coupling at or near the active site. Thus, it would be advantageous to direct coupling to a single position which does not interfere with binding and internalization.

One possibility is to genetically introduce a reactive group, such as a cysteine residue containing a reactive sulfhydryl group, at a defined position, e.g. the N- or C-terminus. This approach was already applied to conjugate single-chain Fv fragments containing an additional C-terminal cysteine residue to liposomal carrier systems (Marty et al., 2002Go; Nielsen et al., 2002Go). However, as in the case of EGF, ligands may contain already several cysteine residues which complicates expression and purification of correctly folded ligands. Furthermore, the free sulfhydryl group leads to dimerization due to cross-linking of two ligands, which requires reduction of the purified ligand under mild conditions prior to coupling.

An alternative approach is the coupling via the N-terminal amino group. This approach is, however, only feasible if the ligand does not contain additional amino groups, i.e. {epsilon} amino groups of lysine residues. Such an approach was realized by the group of Wagner using mouse EGF, which is naturally devoid of lysine residues, for coupling to polyethylenimine/DNA complexes through the bifunctional SPDP cross-linking reagent (Blessing et al., 2001Go). In another study, mouse EGF was used to produce targeted liposomes by activating EGF with Traut’s reagent and coupling to maleimide-PEG lipids (Kullberg et al., 2003Go). However, for therapeutic applications it would be advantageous to use human EGF in order to avoid the induction of a neutralizing human anti-mouse immune response.

In this study, we explored the possibility of generating EGF variants devoid of the two lysine residues present in human EGF. Since the guanidinium group of arginine may also serve as target for amino-reactive coupling groups under certain conditions we included mutagenesis of the three arginine residues of human EGF. Using a limited set of codons, avoiding lysine-encoding triplets but allowing arginine-encoding triplets, we were able to identify a panel of EGF variants lacking all lysine residues and containing only one arginine residue at the original position 41. Such EGF variants should be advantageous for directional and optimized chemical coupling to carrier systems or other molecules.


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

Recombinant human EGF was purchased from Promega (Mannheim, Germany). Fluorescein isothiocyanate (FITC) was purchased from Sigma (Deisenhofen, Germany). S11-EGF DNA was kindly provided by R.Hawkins (Manchester, UK). Lipids were purchased from Avanti (Alabaster, AL, USA) and NHS-PEG2000-DSPE from Shearwater Polymers (now Nektar; Birmingham, AL, USA). Oligonucleotides were purchased from MWG (Ebersberg, Germany)

Oligonucleotides

LMB3, 5'-CAG GAA ACA GCT ATG ACC-3'; LMB2, 5'-GTA AAA CGA CGG CCA GT-3'; fdSeq1, 5'-GAA TTT TCT GTA TGA GG-3'; EGFSfiBack, 5'-TAT GCG GCC CAG CCG GCC ATG GCC AAT AGT GAC TCT GAA TGT-3'; EGFNotFor, 5'-AGT CAG TGC GGC CGC GCG CAG TTC CCA CCA CTT CAG-3'; RGD-4CFor, 5'-AGT TTC TGC GGC CGC CCC GCA GAA ACA ATC ACC CCT ACA GTC GCA GGC CCC GTC TGC CAT GGC CGG CTG GGC CGC ATA-3'; SfiBack, 5'-TAT GCG GCC CAG CCG GCC ATG-3'; NotFor2, 5'-AGT TTC TGC GGC CGC CCC-3'; SfiHisEGF2, 5'-TAT GCG GCC CAG CCG GCC ATG GCC GGA CAT CAC CAT CAT CAC CAT GCG AAT AGT GAC TCT GAA TGT-3'; EGFstopm1, 5'-AGT CAG TGC GGC CGC TTA ACT CAG TTC CCA CCA ACT CAG-3'; K1Back, 5'-ATT GAA GCT TTG GAC BNK TAT GCA TGC AAC TGT GGT-3'; K2For, 5'-CAG TGC GGC GCG ACG CAG TTC CCA CCA MNV CAG GTC TCG GTA CTG ACA-3'; K3For, 5'-CAG TGC GGC CGC ABB CAG TTC CCA CCA ABB CAG GTC ABB GTA CTG ACA ABB CTC CCC GAT GTA GCC AAC-3'; EGFHindFor, 5'-GTC CAA AGC TTC AAT ATA CAT GCA-3'.

Phage display

Human EGF was PCR amplified from clone S11-EGF (Watkins et al., 1997Go) with primers EGFSfiBack and EGFNotFor and cloned into the SfiI/NotI cloning sites of fdVT3 (fdEGF) or pHEN1 (pmEGF). Peptide RGD-4C was amplified from oligonucleotide RGD-4CFor with primers SfiBack and NotFor2 and cloned as SfiI/NotI fragment into fdVT3 (fdRGD-4C). Correct sequences were confirmed by sequencing double-stranded phage DNA with primer fdSeq1. fdEGF and fdRGD were purified from 500 ml bacterial cultures grown overnight at 30°C. pmEGF was obtained by rescue with helper phage as described (Kontermann, 2001Go). Phage were precipitated with 1/5 volume of 20% PEG-6000/5 M NaCl for 1 h on ice. After centrifugation for 15 min at 2500 g, phage were resuspended in 500 µl of PBS.

Library construction

EGF libraries were generated by a two-fragment ligation procedure. Fragment 1 was produced by PCR with primers EGFSfiback and EGFHindFor introducing a HindIII site between codons 24 and 26. Fragment 2 was produced by PCR with primers K1Back and either K2For or K3For, thus introducing randomized codons and a HindIII site. Fragment 1 was digested with SfiI and HindIII and fragment 2 with HindIII and NotI. The two fragments were then cloned into phagemid vector pHEN3 digested with SfiI and NotI.

Cell selections of EGF variants

A431 (5x106 cells) were resuspended in ice-cold DMEM containing 10% FCS, phage were added and incubated for 1 h on ice. Cells were then washed six times with ice-cold medium and once with PBS. The cell pellet was resuspended in 500 µl of 100 mM triethylamine and incubated for 7 min. Cells were pelleted and supernatant was neutralized by adding 250 µl of 1 M Tris–HCl pH 7.4. A 400 µl volume of the phage solution was added to 10 ml of log-phase TG1 and incubated for 60 min at 37°C. Phage were titrated by plating dilutions onto 2xTY, amp, 1% glucose plates. One hundred microlitres of the remaining phage were directly used for a second round of selection.

Labelling of phage with FITC

Phage (~1012–1013 t.u.) were precipitated by adding 1/5 volume of 20% PEG-6000/5 M NaCl and incubation for 10–20 min on ice. After centrifugation for 5 min at 14 000 g, phage were resuspended in 500 µl of 50 mM carbonate buffer pH 9.5. Fifty microlitres of a freshly prepared FITC solution (1 mg/ml in 50 mM carbonate buffer pH 9.5) were added and incubated rotating for 15 min at room temperature in the dark. The reaction was stopped by adding 25 µl of 1 M NH4Cl (final concentration 50 mM) and incubation for another 15 min. Phage were precipitated by adding 1/5 volume of 20% PEG-6000/5 M NaCl and incubation for 10–20 min on ice. After centrifugation for 15 min at 2500 g, the phage pellet was resuspended in 1 ml of ice-cold PBS/PEG/NaCl solution (PBS, 4% PEG-6000, 1 M NaCl) and centrifuged for 2 min at 13 000 r.p.m. This washing step was repeated once. Finally, the phage pellet was resuspended in 250–500 µl of PBS. Approximately 1–10 µl were used for binding studies. Labelled phage were stored in the dark at 4°C.

FACS analysis

Cells (A431, HEK293) were harvested and resuspended in cell culture medium (DMEM) to obtain a cell density of 106 cells/ml. Approximately 105 cells were incubated with phage at the indicated concentration for 30 min at 4°C. Cells were washed twice with PBS, 1% BSA and resuspended in 500 µl of PBS, 1% paraformaldehyde. Binding of phage was analysed with a FACScalibur (Becton-Dickinson, Heidelberg, Germany).

Internalization study

Cells plated onto microscopy cover slips were incubated at 37°C with FITC-labelled phage diluted in cell culture medium to a concentration of 1011–1012 t.u./ml. After 1–4 h cells were washed twice with medium and incubated further at 37°C in medium. Cell staining was analysed by confocal microscopy (Leica TCS 4D).

Expression of recombinant EGF

EGFm1 DNA was amplified with primers SfiHisEGF and EGFstopm1 and cloned as a NcoI/NotI fragment into pET22b (Novagen, Schwalbach, Germany). EGF containing a hexa histidyl tag at the N-terminus was purified from the periplasm by immobilized metal affinity chromatography (IMAC) (Qiagen, Hilden, Germany), as described for recombinant scFv fragments (Kontermann et al., 1997Go). EGF was further purified by FPLC size-exclusion chromatography on a Superose 12 column (Pharmacia, Freiburg, Germany). SDS–PAGE analysis was performed with 20% tricine gels.

Competition experiments

For competition experiments, A431 cells were incubated with 1011 wild-type fdEGF phage and varying concentrations (2.5–50 µg/ml) of purified rhEGF or EGFm1 for 30 min on ice. After washing cells with PBS, bound phage were detected with mouse anti-M13 antibody (Pharmacia) and Cy3-labelled goat-anti-mouse antibody. Binding was analysed by FACS and inhibition of phage binding by EGF was calculated from the mean fluorescence intensity.

Labelling of EGF with FITC

EGFwt or EGFm1 (50 µg) were diluted to a concentration of 500 µg/ml and the pH was adjusted to 9.0 with carbonate buffer pH 9.5. FITC was added at a 10-fold molar excess and incubated overnight at 4°C. Labelled EGF was separated from FITC by gel filtration on Sephadex 25 (Pharmacia). Fluorescence intensity was determined using a fluorescence spectrophotometer (Victor2; Wallac, Freiburg, Germany).

Coupling of EGFm1 to liposomes

Liposomes consisting of neutral phospholipids, cholesterol, NHS-PEG2000-distearoylphosphatidylethanolamine (NHS-PEG2000-DSPE) and rhodamine-dipalmitoyl-phosphatidyl ethanolamine (Rh-DPPE) at a molar ratio of 6:3:1:0.3 were prepared from dried films by hydration with PBS pH 6.0. After extrusion through 50 nm filters, the pH was adjusted to pH 8 with NaOH and EGFm1 was immediately added at a concentration of 50 µg per 1 µmol of lipid. After incubation for 16 h at room temperature, unbound ligands were removed by gel filtration on Sepharose 4B (Pharmacia).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Phage display of human EGF

Amino acids 1–53 of human EGF were displayed on filamentous phage either in a multivalent phage (fdEGF) or monovalent phagemid (pmEGF) format. Both phage preparations showed specific binding to EGFR-expressing A431 cells. In this experiment no significant difference in binding intensity was observed between multivalent and monovalent displayed EGF (data not shown).

The functionality of phage-displayed EGF was further demonstrated by internalization of phage after binding to A431. For this experiment we developed a novel method of applying phage, which were directly labelled with FITC. An analysis of the structure of the major coat protein VIII indicated that ITC-reactive amino groups are provided by the N-terminus (A1) and by the {epsilon} amino group of a lysine residue at position 8 (K8). For labelling with FITC, phage were precipitated, resuspended in 50 mM carbonate buffer pH 9.5 and subsequently incubated with FITC. A time course analysis of labelling fdEGF with FITC showed that efficient labelling was reached already after 5–15 min of incubation. Longer incubation of phage with FITC for up to 1.5 h did not significantly increase labelling (data not shown). Labelling did not reduce transduction efficiency as shown by a comparison of fdEGF before and after labelling with FITC (1.7x1013 versus 1.5x1013 t.u./ml).

Incubation of EGF receptor expressing A431 cells with FITC-labelled fdEGF (fdEGF-FITC) resulted in a strong surface staining of the cells, demonstrating that labelling does not interfere with receptor binding. A detailed analysis of fdEGF-FITC revealed a concentration-dependent staining of A431 cells (Figure 1). Binding could be detected using as little as 109 t.u./ml. Only weak staining at the highest phage concentration was observed with 293 cells, included as a negative control. An FITC-labelled control fd phage displaying an RGD peptide (RGD-4C; Koivunen et al., 1995Go) did not show significant binding to A431 or 293 cells at the highest phage concentration.



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Fig. 1. Binding of FITC-labelled fdEGF phage to EGF receptor-expressing A431 cells. Varying concentrations of fdEGF phage were analysed for binding to A431 or HEK293 cells (0, cells without phage; 1, 1012 t.u.; 2, 1011 t.u.; 3, 1010 t.u.; 4, 109 t.u.). As a control we included FITC-labelled fdRGD phage displaying a cyclic RGD peptide (1012 t.u.).

 
Internalization of phage fdEGF-FITC incubated with A431 at 37°C in medium could be directly demonstrated by confocal immunofluorescence microscopy. Within the first hour, binding of the phage to the cells was seen as filamentous (hairy) structures on the cell surface (Figure 2). Upon further incubation, phage became internalized, resulting in perinuclear accumulation of the particles, which was most prominent after 6–22 h of incubation (Figure 2). No staining was observed with control phage fdRGD-FITC (data not shown). In summary, phage-displayed EGF retains receptor-binding activity and leads to receptor-mediated endocytosis of phage. The direct labelling approach presented here allows direct analysis of phage binding and internalization without the need for secondary detection reagents and further manipulation of the cells.



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Fig. 2. Confocal microscopy of FITC-labelled fdEGF phage incubated with A431 at 37°C. Cells were incubated with phage for 4 h. Cells were then washed and incubated for up to 22 h at 37°C. After 1 h of incubation filamentous structures can be located at the cell surface. With increasing incubation time, phage accumulate within the cells and a perinuclear accumulation becomes visible which is most prominent after 6–22 h of incubation.

 
Selection of lysine-deficient EGF variants

For the selection of lysine-deficient EGF variants we generated two EGF libraries (Figure 3). The first EGF library (EGF1) was randomized at positions K28 and K48 using the triplet BNK coding for 15 amino acids (LVFYWRHDECQSGAP). The second library (EGF2) was randomized at positions K28, R41, R45, K48 and R53 using the triplet BNK for K28 and the triplet VVT coding for nine, mainly hydrophilic, amino acids (RHTSDNGAP) for the remaining positions. Mutagenesis of arginine residues was included since the guanidinium group can serve as target for amino-reactive coupling reagents under certain conditions. Library EGF1 encodes a diversity of 225 possible permutations and library EGF2 of 9.8x104 permutations. Both libraries are devoid of lysine residues but allow arginine at the randomized positions. Both libraries were cloned into phagemid vector pHEN3. Library sizes (positive inserts) were 4x105 for EGF1 and 4.6x104 for EGF2.



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Fig. 3. Sequence of EGF of various species. The positions of lysine and arginine residues are highlighted by grey boxes.

 
Selections were performed on EGF-expressing A431 cells. Phage eluted from the first round were directly used for the second round without any amplification. After two rounds, between 92 and 100% of the eluted phage stained positive in immunofluorescence analysis of cell binding (Table I). The sequence data of several positive clones are summarized in Table II. A preference for hydrophobic residues was observed for phage selected from library EGF1. Only one clone (no. 3) contained an arginine at position 48 instead of lysine (Table II). Most interestingly, all phage selected from the EGF2 library had the original arginine at position 41, indicating that R41 is essential for binding of EGF to its receptor. Only one clone (no. 9) contained the original arginine at position 45 (Table II). In summary, from both libraries we selected several EGF variants devoid of lysine residues. Furthermore, the number of arginine residues was reduced from three to one in most of the EGF variants selected from library EGF2 (see also Figure 4A for summary).


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Table I. Cell selection of EGF variants
 

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Table II. Amino acids at randomized positions of clones isolated from EGF libraries 1 and 2
 


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Fig. 4. (A) Three-dimensional structure of EGF (Cooke et al., 1987Go) visualized with RasMac 2.6 (Roger Sayle, Glaxo Wellcome, UK) with the positions of lysines and arginines marked. The corresponding amino acids identified from the EGF libraries are indicated. (B) FACS analysis of fdEGFwt and the two EGF variants fdEGFm1 and fdEGFm2 for binding to A4331 cells.

 
For further analysis we selected two EGF variants: EGFm1 (clone 6 of the EGF2 library selection) containing hydrophilic residues and EGFm2 (clone 10 of the EGF2 library selection) containing hydrophilic and small aliphatic residues at the randomized positions (see Table II). No differences in binding of 5x1010 EGFwt, EGFm1 or EGFm2 phage to A431 cells was observed by FACS analysis (Figure 4B). This finding indicates that EGF variants bind with the same affinity to the EGF receptor as EGFwt.

Expression of recombinant EGF variants

Since no differences in binding of EGFm1 or EGFm2 phage were observed we selected EGFm1 for soluble expression. EGFm1, containing a hexahistidyl tag at its N-terminus, was purified by IMAC in soluble form from the periplasm of transformed BL21 DE3 cells. Approximately 0.6 mg was purified from 1 l of bacterial culture induced for 3 h at 23°C. SDS–PAGE analysis confirmed the correct size of 7 kDa (Figure 5A). As expected, EGFm1 migrated slightly slower than recombinant EGFwt lacking the hexahistidyl tag. This was further confirmed by size-exclusion chromatography (Figure 5B). This experiment also revealed the presence of molecules with a size of ~15 kDa in the EGFm1 preparation corresponding in size to dimeric EGF molecules.



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Fig. 5. (A) IMAC purification of recombinant HisEGFm1 from bacterial periplasm. Eluted fractions were analysed on a 20% SDS–tricine gel (HisEGFm1 is indicated by an arrowhead). Recombinant human EGF was included as control. (B) Size-exclusion chromatography of purified EGFwt and HisEGFm1 on a Superose 12 column. The positions of BSA (67 kDa), ovalbumin (43 kDa) and myoglobin (17 kDa) are indicated by arrows. (C) Competition of binding of fdEGF to A431 by EGFwt or HisEGFm1.

 
Binding of purified EGFm1 to the EGF receptor was demonstrated by competition of binding of EGFwt-displaying phage to A431 cells by soluble EGFm1 or EGFwt (Figure 5C). A titration of EGFm1 compared with EGFwt gave identical values with an IC50 of ~0.5 µM indicating that EGFm1 binds with the same affinity as EGFwt to A431 cells.

Coupling of amino-reactive reagents to EGFm1

Reactivity of EGFm1 with amino-reactive reagents was demonstrated with FITC. EGFm1 or EGFwt were incubated with excess amounts of FITC and separated by gel filtration. The protein peak fractions of EGFwt had a fluorescence intensity of 84 945 units compared with 29 720 units for EGFm1. Thus, the ratio of labelling of EGFwt to EGFm1 was 2.9, which is almost identical to the 3:1 ratio of amino groups present in these proteins. Fluorescein-labelled EGFm1 showed strong and concentration-dependent binding to A431 cells, demonstrating that labelling did not interfere with binding to the EGF receptor (Figure 6A). In this experiment, binding of fluorescein-labelled EGFm1 to A431 reached saturation at ~50 ng/ml and could be competed with excess amounts of unlabelled EGFwt but not with BSA (Figure 6B and C). In these experiments we also observed binding of EGFwt to A431 cells to a certain extent, indicating that labelling of EGFwt with FITC still allows binding to the EGF receptor (data not shown).



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Fig. 6. Binding of FITC-labelled EGFm1 to A431. (A) FITC-labelled HisEGFm1 was incubated with A431 at varying concentrations and binding was analysed by flow cytometry (0, cells without EGFm1; 1, 17 ng/ml; 2, 50 ng/ml; 3, 170 ng/ml). (B) Competition of FITC-labelled EGFm1 at 170 ng/ml without (1) or with (2) excess amounts of unlabelled EGFwt. (C) Control experiments with BSA.

 
Binding of EGFm1 liposomes to A431

In a further experiment we coupled EGFm1 to rhodamine-labelled liposomes containing 10 mol% NHS-PEG2000-DSPE. Thus, these liposomes display EGFm1 at the end of the PEG chain. Incubation with A431 cells demonstrated a strong binding to these cells compared with the same liposomes lacking EGFm1 (Figure 7A). In contrast, no binding was observed with EGFR-negative 293 cells (Figure 7B). As observed before for FITC-labelled EGFm1, binding to A431 cells could be completely inhibited by excess amounts of EGFwt but not with BSA (Figure 7C and D). Incubation of A431 cells with EGFm1 liposomes at 37°C resulted in internalization as evidenced by a perinuclear accumulation of fluorescence (Figure 7E). No such pattern was observed on incubating A431 cells at 4°C (data not shown). In addition, no staining and internalization were seen with unconjugated liposomes or using 293 cells (data not shown).



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Fig. 7. (A) Binding of EGFm1 liposomes to A431 cells. EGFm1 liposomes (2) showed specific binding to A431 cells, in contrast to unconjugated liposomes (1), which did not show binding to A431 cells (0). No binding was observed with 293 cells (B). Excess amounts of EGFwt completely inhibited binding of EGFm1 liposomes to A431 cells (C), in contrast to BSA (D) which did not show any effects (0, cells alone; 1, EGFm1 liposomes; 2, EGFm1 liposomes + EGFwt or BSA. (E) Internalization study incubation of A43 cells with EGFm1 liposomes for 6 h at 37°C (left, fluorescence microscopy image; right, phase-contrast image). Internalization is evidenced by a perinuclear accumulation of fluorescence.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Applying phage-displayed libraries of EGF we were able to select EGF variants with full binding activity which are deficient of lysine residues and which lack two of the three arginine residues. A diverse set of residues was found at positions 28, 45, 48 and 53. We did not observe a bias towards a conservative substitution of the lysine residues by arginine residues. This is in accordance with previous findings, which showed that the electrostatic property of K28 and K48 is not required for receptor–ligand association (Campion et al., 1992Go). Our results also confirmed the importance of arginine 41 for receptor binding of EGF established previously by site-directed mutagenesis and chemical modification experiments (Engler et al., 1992Go). This study revealed that even the charge-conservative substitution by lysine leads to inactive EGF molecules. This is also in accordance with a previous study using a library of EGF with a randomized position 41 which resulted only in the isolation of EGF molecules containing the wild-type arginine residue (Souriau et al., 1997Go).

Using FITC we could demonstrate that EGFm1 can be conjugated to an amino-reactive reagent. Hence, the EGF variants generated in our study should allow for the site-specific conjugation to drugs or drug carrier systems employing the N-terminal amino group. Studies with genetically engineered EGF fusion proteins already demonstrated that fusion of EGF via its N-terminus to other molecules does not interfere with receptor binding (Watkins et al., 1997Go). This was also confirmed by studies employing mouse EGF which was coupled via the N-terminus to either PEI/DNA complexes or to liposomes (Blessing et al., 2001Go; Kullberg et al., 2003Go). However, since mouse EGF is only 70% homologous to human EGF it can be expected that conjugates containing mouse EGF will lead to an immune response in humans. Consequently, as observed for therapeutic murine antibodies, repeated injections will result in neutralization and thus in a reduced or even abolished therapeutic efficacy. Our human EGF variants should be able to circumvent this limitation, although we cannot exclude that the four amino acid substitutions introduced by us into EGF will also cause immunogenicity in humans. Nevertheless, our EGF variants should avoid the often observed ligand-mediated cross-linking and aggregate formation of drug carrier systems, such as liposomes or other polymers, which prevents the coupling of ligands at high densities. In preliminary experiments we could demonstrate that EGFm1 can be used to generate liposomal carrier systems targeting EGF receptor-expressing cells, which are efficiently endocytosed by the target cells.

Since natural ligands from other species which are devoid of lysines are not always available and since ligands from other species may also exhibit reduced affinity or altered specificity for human receptors, this approach might be a valuable tool for the generation of lysine-deficient human ligands. Indeed, a similar approach was recently applied to generate lysine-deficient TNF-{alpha} variants (Yamamoto et al., 2003Go). In this study the six lysine positions were randomized using the triplet NNS and TNF-{alpha} variants were selected against a TNF-{alpha} neutralizing antibody or TNF-RI. Interestingly, although two lysine residues were described to be vital for bioactivity, several of the TNF-{alpha} variants had other residues at these positions. One of the lysine-deficient TNF-{alpha} variants with full biological activity could be specifically mono-PEGylated at its N-terminus leading to improved antitumour activity compared with randomly mono-PEGylated wild-type TNF-{alpha}. This study clearly demonstrates the usefulness of lysine-deficient ligands for site-directed modifications to improve therapeutic efficacy and further shows that phage display represents a powerful tool to isolate novel biologically active proteins with the desired properties. As described in our study, the usage of triplets which do not encode for lysines should facilitate the enrichment of lysine-deficient active ligands.


    Acknowledgements
 
The technical assistance of Margitta Alt is gratefully acknowledged. We thank Christina Schön for help in the production of recombinant EGFm1 and Abdo Konur for help with FACS analysis.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aboud-Pirak,E., Hurwitz,E., Bellot,F., Schlessinger,J. and Sela,M. (1989) Proc. Natl Acad. Sci. USA, 86, 3778–3781.[Abstract]

Allen,T.M. (2002) Nat. Rev., 2, 750–763.[CrossRef][ISI]

Baselga,J. (2001) J. Clin. Oncol., 19, S41–S44.[ISI][Medline]

Blessing,T., Kursa,M., Holzhauser,R. Kircheis,R. and Wagner,E. (2001) Bioconjug. Chem., 12, 529–537.[CrossRef][ISI][Medline]

Campion,S.R., Tadaki,D.K. and Niyogi,S.K. (1992) J. Cell. Biochem., 50, 35–42.[ISI][Medline]

Carpenter,G. and Cohen,S. (1990) J. Biol. Chem., 265, 7709–7712.[Free Full Text]

Chen,P., Mrkobrada,M., Vallis,K.A., Cameron,R., Sandhu,J., Hendler,A. and Reilly,R.M. (2002) Nucl. Med. Biol., 29, 693–699.[CrossRef][ISI][Medline]

Ciardiello,F. and Tortora,G. (2001) Clin. Cancer Res., 7, 2958–2970.[Abstract/Free Full Text]

Cooke,R.M., Wilkinson,A.J., Baron,M., Pastore,A., Tappin,M.J., Campbell,I.D., Gregory,H. and Sheard,B. (1987) Nature, 327, 339–341.[CrossRef][ISI][Medline]

Engler,D.A., Campion,S.R., Hauser,M.R., Cook,J.S. and Niyogi,S.K. (1992) J. Biol. Chem., 267, 2274–2281.[Abstract/Free Full Text]

Forssen,E. and Willis,M. (1998) Adv. Drug Deliv. Rev., 29, 249–271.[CrossRef][ISI][Medline]

Jinno,H., Ueda,M., Ozawa,S., Kikuchi,K., Ikeda,T., Enomoto,K. and Kitajima,M. (1996) Cancer Chemother. Pharmacol., 38, 303–308.[CrossRef][ISI][Medline]

Kikuchi,A., Sugaya,S., Ueda,H., Tanaka,K., Aramaki,Y., Hara,T., Arima,H., Tsuchiya,S. and Fuwa,T. (1996) Biochem. Biophys. Res. Commun., 227, 666–671.[CrossRef][ISI][Medline]

Koivunen,E., Wang,B. and Ruoaslahti,E. (1995) Biotechnology, 13, 265–270.[ISI][Medline]

Kontermann,R.E. (2001) In Kontermann,R.E. and Dübel,S. (eds), Antibody Engineering. Springer, Heidelberg, pp. 137–148.

Kontermann,R.E., Martineau,P., Cummings,C.E., Karpas,A., Allen,D., Derbyshire,E. and Winter,G. (1997) Immunotechnology, 3, 137–144.[CrossRef][ISI][Medline]

Kullberg,B.E., Bergstrand,N., Carsson,J., Edwards,K., Johnsson,M., Sjoberg,S. and Gedda,L. (2002) Bioconjug. Chem., 13, 737–743.[CrossRef][ISI][Medline]

Kullberg,E.B., Nestor,M. and Gedda,L. (2003) Pharm. Res., 20, 229–236.[CrossRef][ISI][Medline]

Lee,C.H., Lee,E.C., Tsai,S.T., Kung,H.J., Liu,Y.C. and Hwang,J. (1993) Protein Eng., 6, 433–440.[ISI][Medline]

Lemmon,M.A., Bu,Z., Ladbury,J.E., Zhou,M., Pinchasi,D., Lax,I., Engelman,D.M. and Schlessinger,J. (1997) EMBO J., 16, 281–294.[Abstract/Free Full Text]

Lutsenko,S.V., Feldman,N.B., Finakova,G.V., Posypanova,G.A., Severin,S.E., Skryabin,K.G., Kirpichnikov,M.P., Lukyanets,E.A. and Vorozhtsov,G.N. (1999) Tumor Biol., 20, 218–224.[CrossRef][ISI]

Lutsenko,S.V., Feldman,N.B., Finakova,G.V., Gukasova,N.V., Petukhov,S.P., Posypanova,G.A., Skryabin,K.G. and Severin,S.E. (2000) Tumor Biol., 21, 367–374.[CrossRef][ISI]

Lutsenko,S.V., Feldman,N.B. and Severin,S.E. (2002) J. Drug Target, 10, 567–571.[ISI][Medline]

Marty,C., Odermatt,B., Schott,H., Neri,D., Ballmer-Hofer,K., Klemenz,R. and Schwendener,R.A. (2002) Br. J. Cancer, 87, 106–112.[CrossRef][ISI][Medline]

Nicholson,R.I., Gee,J.M.W. and Harper,M.E. (2001) Eur. J. Cancer, 37, S9–S15.[ISI][Medline]

Nielsen,U.B., Kirpotin,D.B., Pickering,E.M., Hong,K., Park,J.W., Shalaby,M.R., Shao,Y., Benz,C.C. and Marks,J.D. (2002) Biochim. Biophys. Acta, 1591, 109–118.[CrossRef][ISI][Medline]

Park,J.W. et al. (2002) Clin. Cancer Res., 8, 1172–1181.[Abstract/Free Full Text]

Salomon,D.S., Bradt,R., Ciardiello,F. and Normanno,N. (1995) Crit. Rev. Oncol. Hematol., 19, 183–232.[CrossRef][ISI][Medline]

Schmidt,M., Vakalopoulou,E., Schneider,D.W. and Wels,W. (1997) Br. J. Cancer, 75, 1575–1584.[ISI][Medline]

Souriau,C., Fort,P., Roux,P., Hartley,O., Lefranc,M.-P. and Weill,M. (1997) Nucleic Acids Res., 25, 1585–1590.[Abstract/Free Full Text]

Watkins,S.J., Mesyanzhinov,V.V., Kurochkina,L.P. and Hawkins,R.E. (1997) Gene Ther., 4, 1004–1012.[CrossRef][ISI][Medline]

Yamamoto,Y. et al. (2003) Nat. Biotechnol., 21, 546–551.[CrossRef][ISI][Medline]

Received July 8, 2003; revised October 23, 2003; accepted October 30, 2003





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