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
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Abstract |
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Keywords: EGF receptor/epidermal growth factor/liposomes/mutagenesis/phage display/targeting
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Introduction |
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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, 1990; Lemmon et al., 1997
). 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., 1995
; Baselga, 2001
; Ciardiello and Tortora, 2001
; Nicholson et al., 2001
). 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., 1993
; Jinno et al., 1996
; Lutsenko et al., 1999
, 2000
, 2002
; Chen et al., 2002
). Furthermore, liposomes and viral vectors displaying EGF on their surface were developed for target cell-specific drug or gene delivery (Kikuchi et al., 1996
; Kullberg et al., 2002
, 2003
). 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., 1989
; Schmidt et al., 1997
).
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., 2002; Nielsen et al., 2002
). 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. 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., 2001
). In another study, mouse EGF was used to produce targeted liposomes by activating EGF with Trauts reagent and coupling to maleimide-PEG lipids (Kullberg et al., 2003
). 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.
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Materials and methods |
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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., 1997) 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, 2001
). 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 TrisHCl 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 (10121013 t.u.) were precipitated by adding 1/5 volume of 20% PEG-6000/5 M NaCl and incubation for 1020 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 1020 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 250500 µl of PBS. Approximately 110 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 10111012 t.u./ml. After 14 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., 1997). EGF was further purified by FPLC size-exclusion chromatography on a Superose 12 column (Pharmacia, Freiburg, Germany). SDSPAGE 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 (2.550 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).
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Results |
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Amino acids 153 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 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 515 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., 1995) did not show significant binding to A431 or 293 cells at the highest phage concentration.
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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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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. SDSPAGE 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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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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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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Discussion |
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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., 1997). 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., 2001
; Kullberg et al., 2003
). 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- variants (Yamamoto et al., 2003
). In this study the six lysine positions were randomized using the triplet NNS and TNF-
variants were selected against a TNF-
neutralizing antibody or TNF-RI. Interestingly, although two lysine residues were described to be vital for bioactivity, several of the TNF-
variants had other residues at these positions. One of the lysine-deficient TNF-
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-
. 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.
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Acknowledgements |
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References |
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Received July 8, 2003; revised October 23, 2003; accepted October 30, 2003