Bioconjugate Chem. 1995, 6,683-690
683
A New Radioligand for the Epidermal Growth Factor Receptor: lllIn Labeled Human Epidermal Growth Factor Derivatized with a Bifunctional Metal-Chelating Peptide Sandrine RBmy,' Raymond M. Reilly,* Katherine Sheldon,g a n d J e a n GariBpy",' Department of Medical Biophysics, University of Toronto, and The Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada M5G 2C1. Received May 15, 1 9 9 P
More specific radiopharmaceuticals are currently being evaluated for the in vivo detection and therapy of breast cancer. The human epidermal growth factor (hEGF) represents a good radiopharmaceutical candidate in view of the reported overexpression of its receptor by breast cancer cells. To enhance the imaging potential of this peptide ligand, a synthetic strategy was developed to rapidly create small peptides containing a large number of metal-chelating groups that can be readily coupled to hEGF. A prototypic 15-amino acid branched peptide containing four EDTA-like chelator groups was assembled by solid phase peptide synthesis. The metal chelating peptide, abbreviated MCP-4-EDTASH, was selectively incorporated into hEGF(1-51) a t its unique N-terminus amino group. The coupling of a single MCP-4-EDTA-SH into hEGF(1-51) was confirmed by SDS polyacrylamide gel electrophoresis, western blotting, and amino acid analysis. The protein conjugate was successfully labeled with IllIn. Its specific binding to EGF receptors present on MDA-MB-468breast cancer cells confirmed that such a construct retains the properties of the natural ligand.
INTRODUCTION
Breast cancer represents a major cause of mortality in North American women with more than 40 000 deaths reported in 1994 alone ( I ) . The epithelial cells of the breast are under the influence of a variety of hormones (estrogens, progestins, prolactin) and growth factors. An aberration in the hormonal milieu has been postulated to be one of the critical factors in the development of breast cancer ( 2 , 3 ) . Numerous studies have suggested that the level of expression of epidermal growth factor receptors (EGFRs) may represent a useful prognostic factor in breast cancer. The overexpression of EGFRs has been observed in many primary breast cancer tissues as well as in established breast tumor cell lines. It has been associated with a poor response to endocrine therapy with tamoxifen, a predilection to recurrent disease, and a decreased long-term survival ( 4 ) . EGFR may thus be an attractive target for the design of imaging and therapeutic probes (5, 6 ) . Radiolabeled monoclonal antibodies (MAbs) have been used to develop specific radiopharmaceuticals for imaging and for therapeutic strategies for breast cancer (7). However, the antigens targeted in past studies have not been agents that influence the progression of the disease. Radiolabeled MAbs against EGFR are presently being evaluated in terms of their ability to localize into solid tumors. lz3IEGFRl mAb and IZ5I-anti-EGFR-425have been used to image or treat brain gliomas in patients (8,9),to image RT4 human bladder tumor xenografts in nude mice (IO), while lllIn-mAb 225 is under investigation in phase I therapy and imaging trials in patients with squamous ~~~
~
~~
* Author to whom correspondence should be addressed. Phone: (416) 946-2967. Fax: (416) 946-6529. E-mail: gariepy@ ocic1.oci.utoronto.ca. + Department of Medical Biophysics, Ontario Cancer Institute. Division of Nuclear Medicine, The Toronto Hospital, and Faculty of Pharmacy, University of Toronto, Ontario, Canada. Present address: Boston Biomedical Research Institute, 20 Stanisford St., Boston, MA 02114. Abstract published in Advance ACS Abstracts, September 15, 1995. @
cell lung carcinoma (11). The main problems with targeting approaches involving the use of monoclonal antibodies are associated with their low uptake by the tumor, slow elimination from the blood, and the production of human anti-mouse antibodies (HAMA) (12). The alternative approach of using the natural ligand, epidermal growth factor (EGF) itself, remains somewhat unexplored. One imaging study reported the use of lZ3IEGF in patients with advanced cervical cancer, with a high tumorlnontumor ratio, 6-8 h after injection (13). Another study demonstrated imaging of glioma tumors in rats following intracerebral injection of 99mTc-EGF (14). In terms of developing powerful radiolabeled imaging agents involving the use of protein-based targeting agents such as EGF, one is faced with the practical compromise of introducing a sufficient number of individual metalchelating sites on such proteins to generate conjugates that are radiochemically useful without a complete loss of binding affinity of the modified targeting agent for its receptor (as a result of excessive labeling) (15). A simple strategy was thus developed based on the flexibility and rapidity of solid-phase techniques to design bifunctional branched peptides that incorporate a defined number of metal-chelating sites and possess a single reactive site that allows the peptide to be coupled unidirectionally to one designated site available on EGF. We report the chemical and biological properties of a well-defined human EGF(1-51) construct that includes a branched peptide containing four metal-chelating sites, which can be radiolabeled with "'In to a high specific activity and can bind to the EGFR-expressing breast tumor cell line, MDA-MB-468. EXPERIMENTAL PROCEDURES
Chemicals. Human EGF(1-51) was a generous gift from Dr. Maratea (Creative BioMolecules, Hopkinton, MA). All Boc-protected amino acids were purchased from Nova Biochem (LaJolla, CA) and the MBHA resin from Applied Biosystems (Mississauga, Ontario, Canada). Trifluoroacetic acid (TFA, sequencing grade), a,p-diaminopropionic acid, and diisopropylethylamine (DIEA)were
1 043-1 802/95/2906-0683$09.00/00 1995 American Chemical Society
684 Bioconjugate Chem., Vol. 6, No. 6, 1995
purchased from Schweizerhall, Inc. (South Plainfield, NJ). tert-Butyl bromoacetate was obtained from Aldrich Chemical Co. (Milwaukee, WI). Sodium thiophenoxide was prepared by addition of thiophenol to 1molar equiv of sodium metal in diethyl ether. The suspension was vigorously shaken for 72 h at room temperature, and the resulting sodium thiophenoxide was recovered by filtration (16). All reagent grade solvents and chemicals (acetonitrile, dichloromethane (DCM), dicyclohexylcarbodiimide (DCC),N-hydroxybenzotriazole (HOBt), p-mercaptoethanol, sodium borohydride, and 5,5'-dithiobis(2nitrobenzoic acid) (Ellman's reagent (DTNB)) were purchased from BDH (Toronto,Ontario, Canada). 1111nC13 ('50 mCi/mL) was obtained from Nordion (Kanata, Ontario, Canada). NalZ5I(100 mCi/mL) was obtained from NENDupont (Boston, MA) or Amersham (Oakville, Ontario, Canada). Materials and Equipment. lH-NMR spectra were recorded a t 500 MHz on a Brucker AM-500 spectrometer and resonance assignments reported in ppm downfield from the internal tetramethylsilane standard. Mass spectra (MS) were recorded on a VG Masslab 20-250 quadrapole spectrometer and U V spectra on a Beckman DU-30 spectrophotometer. Amino acid analyses were performed on a Beckman System Gold 166 analyzer after the samples had been hydrolyzed in vacuo for 24 h a t 110 "C, in the presence of 6 N constant boiling HC1 containing 0.1% (v/v) phenol. The branched peptide was synthesized on an Applied Biosystems Model 430 peptide synthesizer. Chromatography grade silica gel (200-425 mesh) was purchased from Fisher Scientific. Thin-layer chromatography (TLC) was performed using Whatman silica gel 60 A fluorescent indicator plates. The extent of labeling of peptides with lllIn or lZ5Iwas monitored by instant thin-layer chromatography on ITLC SG silica gel impregnated glass fiber sheets from Gelman Sciences (Ann Arbor, MI). Cell Lines and Culture Conditions. The MDA-MB468 breast cancer cell line (17)was obtained from Dr. Ron Buick (Ontario Cancer Institute, Toronto, Ontario) and routinely cultured in L-15 medium supplemented with 10% fetal calf serum (FCS) (18). EDTA-like Chelator Synthesis. The protected form of the chelator EDTA was prepared following a simple two-step procedure described by Arya and Garibpy (19). Typically, a,@-diaminopropionicacid was refluxed for 20 h in redistilled acetonitrile in the presence of 10 molar equiv of DIEA and 7 molar equiv of tert-butyl bromoacetate. The reaction mixture was precipitated by adding toluene and filtered, and the filtrate was reduced to dryness. The resulting oily mixture was redissolved in toluene and extracted with 0.1 M phosphate buffer, pH 2.0. The organic phases were dried over MgS04, filtered, and evaporated. A flash chromatography step of the crude mixture loaded on a silica gel column provided the pure [(a,/3,N,N-tetra-tert-butylcarboxy)methylldiaminopropionic (tert-buty1carboxy)methyl ester in 60-80% yield. The eluent was 5% v/v acetone in chloroform: MS (FAB) MH' 675; lH-NMR (CDC13) 1.43-1.49 (m, 45 H, t-Bu), 3.14 (2 q, 2 H, NCHCHZN,J H a - H b = 6 HZ, J H ~ - H V 8 Hz, JH~-HV 13.9 Hz), 3.5-3.6 (2 S, 8 H, NCH2COOtBu), 3.77 (9, 1H, NCHCHzN, JHI-HS = 6 Hz, JHI-HP' =8 Hz), 4.46-4.55 (2 d, 2 H, JH-W 15.5 Hz, -O(O)CCHzC(O)O-). The intermediate ester was converted to the protected form of the chelating agent that includes a single free carboxylic arm by reaction with 3-4 molar equiv of sodium thiophenoxide (16)in DMF a t 100 "C for 2 h. The reaction mixture was precipitated by adding ethyl acetate and filtered, and the filtrate was reduced to dryness. The
Remy et al.
resulting mixture, redissolved in ethyl acetate, was extracted with 0.1 M phosphate buffer, pH 2.0, and the organic phases were evaporated. The crude mixture was loaded on a silica gel column, and pure [(a,P,N,N-tetratert-butylcarboxy)methyl]diaminopropionicacid (tert-butyl protected EDTA-like groups) was recovered in 60% yield by flash chromatography. The eluent was 100% toluene to remove unreacted diaminopropionic tert-butyl ester followed by 2% v/v acetic acid in acetone: MS (FAB) MH+ 561; IH-NMR (CDC13) 1.45-1.47 (m, 36 H, t-Bu), 3.12 (d, 2 H, NCHCH$, J H a - H b = JH~-HW = 7.5 Hz), 3.43.6 (m, 8 H, NCH2COOt-Bu), 3.77 (t, 1 H, NCHCHzN). Peptide Synthesis. The metal-chelating peptide MCP-4-EDTA-SH(Figure 1)was prepared by solid-phase peptide synthesis using N-tert-butoxycarbonyl (Boclprotected amino acids and MBHA resin support. The substitution on the resin support was 0.1 mmoVg of resin. The initial low substitution value on the resin ensures that crowding of the support with peptide chains will not occur as a result of two branching steps. Four branches were introduced in the peptide using two successive rounds of addition of (Na, NE) di-Boc-L-lysine. All couplings were carried out using symmetric anhydride derivatives of protected amino acids employing protocols established by the manufacturer (Applied Biosystems, Foster City, CAI. Each coupling step was monitored by the quantitative determination of free amino group present on the resin (quantitative ninhydrin test). The efficiency of each coupling step was greater than 99% (20). The tert-butyl-protected EDTA-like chelator (2 molar equiv in relation to the number of moles of free amino groups in the reaction vessel) was coupled to each of the four amino termini on the branched peptide, in the presence of 2 molar equiv of DCC and HOBt dissolved in 40% DMF/DCM. The slurry was mixed a t room temperature for 16 h. The resin was then washed with DMF, and the coupling step was repeated. The completion of this step was monitored by quantitative ninhydrin and found to be greater than 99%. Any remaining free amino groups were acetylated for 15 min a t room temperature with 10% (v/v) acetyl anhydride, 5% (v/v) DIEA prepared in DCM. The tert-butyl groups protecting the EDTA-like carboxylic groups were cleaved in 25% T F N DCM for 2 h prior to chain detachment. Cleavage, Recovery, and Analysis of MCP-4-EDTASH. The polymer was cleaved from the resin support with 10% (v/v) anisole, 10% (v/v) dimethyl sulfide, and 3% (v/v) thiocresol in anhydrous hydrogen fluoride a t 0 "C for 90 min. The resin was extracted with several ether washes to remove scavengers and cleaved protecting groups. The branched peptide was then recovered by extracting the resin with 100% TFA. The resulting solution was reduced by rotary evaporation in a silanized flask to 2 mL and transferred to a polypropylene tube. The peptide was then precipitated by adding ice-cold ether (30 mL) to the tube and recovered by centrifugation. This cycle was repeated twice, and then the pellet was redissolved in water and lyophilized. The peptide was desalted on a BioGel P-2 column equilibrated with 50 mM ammonium bicarbonate. Fractions migrating with the column void volume were pooled and lyophilized. The composition of the branched peptide was confirmed by amino acid analysis performed on duplicate samples: Ala (1)found 1.2 f 0.1; aminocaproic acid (5) found 4.6 f 0.5; Gly (4) found 3.9 f 0.1; Lys (3) found 2.7 f 0.1; Tyr (1) found 1.3 f 0.1. A peptide-resin sample was recovered and cleaved prior to the coupling of EDTA groups. The resulting peptide (lacking the EDTA groups) was analyzed by ion-spray mass spectrometry and shown to have the expected mass (MH+, 1532). The presence
Bioconjugate Chem., Vol. 6, No. 6, 1995 685
Metal Binding Peptide-EGF Conjugate 3. Branched Lyrlm Polymer Allows for the dwMlng ol free amino groups available on the polymer a1 each coupling during synthesis.
I 4.
vnsl(11e meiml-ehdallng ill*
e site for undirectmal ing 10 targeting agent
Figure 1. Structure of MCP-4-EDTA-SH, a heterobifunctional metal-chelating peptide that incorporates four EDTA groups and a single thiol moiety. Symbols: ACA, aminocaproic acid; Ala, alanine; Cys, cysteine; EDTA, EDTA-like chelator; Gly, glycine; Lys, lysine; Tyr, tyrosine. of 4 EDTA (3.6 f 0.2) groups was confirmed using the colorimetric procedure developed by Darbey (211. Reduction of MCP-CEDTA-SH. The presence of free sulfhydryl groups was quantified using a DTNB assay (22). The single thiol group on the peptide was reduced using sodium borohydride (23). Typically, 100 pL of a freshly prepared solution of sodium borohydride (0.1 MI in cold 0.1 M sodium borate buffer (pH 9.1) was dispensed into a tube containing 1 mg of the peptide MCP-4-EDTA-SH (0.36 pmol) dissolved in 1mL of borate buffer. The reduction reaction was allowed to proceed on ice for 15 min. The solution was then acidified to pH 2.0 by dropwise addition of 6 N HC1 in order to destroy the excess sodium borohydride. The pH of the reaction mixture was then adjusted to pH 6.0 with 4 N NaOH. The reduced peptide was then immediately mixed with the maleimide derivative of hEGF(1-51) to initiate the coupling reaction. Maleimide Derivative of hEGF(1-51). The single amino group of hEGF(1-51) (N-terminus) was derivatized with the bifunctional crosslinking agent, (m-maleimidobenzoy1)-N-hydroxysulfosuccinimide ester (sulfoMBS). Briefly, solid sulfo-MBS (2 mg; 4.6 pmol) was added to hEGF(1-51) (0.5 mg; 86.2 nmol) dissolved in 1 mL of PBS. The reaction was left to proceed a t room temperature for 2 h with continuous agitation. The maleimide derivative abbreviated MB-hEGF(1-51) was desalted from excess unreacted sulfo-MBS by gel filtration on a BioGel P-6 column (1cm x 10 cm) eluted with 0.1 M phosphate buffer pH 6.0. Coupling of MCP-CEDTA-SHto MB-hEGF(1-51). A freshly prepared solution of reduced MCP-4-EDTA-SH peptide (4-fold excess in relation to the number of moles of MB-hEGF(1-51)) was reacted with MB-hEGF(1-51) a t 4 "C for 16 h. The conjugate was purified from unreacted MCP-4-EDTA-SH peptide by gel filtration on a BioGel P-30 column (1 x 10 cm) equilibrated with 0.1 M ammonium bicarbonate (pretreated on Chelex-100 resin). Fractions from the P-30 column were analyzed by ELISA as described above to detect the presence of hEGF( 1-51) containing species. The conjugate eluted in the void volume peak, resolved from MB-hEGF(1-511, and the branched peptide (MCP-4-EDTA-SH). The coupling of a single MCP-4-EDTA-SH peptide to hEGF(151) was confirmed by comparing the ratio of amino acid present in the conjugate but absent in hEGF(1-51). Amino acid analyses were performed on triplicate
@y-!d O
=
Q
+
HS
cys
H
BP
ACA
O
H
lb 0 e . q
cys
H
ACA
5P
H
Figure 2. Conjugation scheme of MCP-4-EDTA-SH to hEGF(1-51). Step a: reaction of N-terminus amino group of hEGF(1-51) with sulfo-MBS. Step b: reaction of the maleimide group on MB-hEGF(1-51) with the free thiol group present on MCP4-EDTA-SH. BP represents the branched peptide defined in Figure 1. Conjugation conditions are described in the Experimental Procedures.
samples: Ala (1)found 0.9 f 0.2; aminocaproic acid ( 5 ) found 4.5 f 0.2; Lys (3) found 2.5 f 0.3. The chelating properties of MCP-4-EDTA-S-MB-hEGF(1-51) conjugate were confirmed by the binding of indium-111 (Figure 5). Polyacrylamide Gel Electrophoresis. The conjugation of MCP-4-EDTA-SHto the protein MB-hEGF(151) was monitored by SDS polyacrylamide gel electrophoresis on 16% Tris-tricine gels following the method of Schagger and von Jagow (24) using a Mini-Protean I1 electrophoresis chamber (Bio-Rad Hercules, CA). Each protein sample was diluted 1 in 4 with sample buffer containing 2% /3-mercaptoethanol and 2% (w/v) Coomassie Blue G-250 and heated a t 95 "C for 4 min before sample loading. The electrophoresis step was performed a t 100 V (constant voltage), with a typical running time of 90 min. The gel was fixed with 10% acetic acid, 40% methanol in water for 0.5 h, stained with Coomassie Blue G-250 for 1h, and finally destained in 10% acetic acid in water.
686 Bioconjugate Chem., Vol. 6,No. 6, 1995
RBmy et al.
Figure 3. Primary structure of the MCP-4-EDTA-S-MB-hEGF(1-51) conjugate. Symbols: A, maleimidobenzoyl linker; ACA, amino caproic acid; EDTA, EDTA-like chelator. Three-letter codes were used for all amino acids.
Generation of Antisera against hEGF(1-51). New Zealand White female rabbits (2.5 kg) were immunized with four subcutaneous injections (0.25 musite) of a 1:l mixture of hEGF(1-51) (250 pg) emulsified in sterile phosphate-buffered saline (PBS; 0.5 mL) and Freund's complete adjuvant (0.5 mL; Sigma Chemical Co., St. Louis, MO). The rabbits were boosted with four subcutaneous injections (0.25 musite) of a 1:l mixture of hEGF(1-51) (250 pg) emulsified in 0.5 mL of PBS and 0.5 mL of Freund's incomplete adjuvant. Antisera titers toward hEGF(1-51) were tested by ELISA. ELISA Assay. Purified hEGF(1-51) or hEGF(1-51) conjugate were used to coat microtiter wells (1pglwell) for 2 h. After four washes with PBS, wells were incubated with 2% (w/v) bovine serum albumin (BSA)in PBS for 1 h. Unbound protein was removed by washing the wells with PBS. The antigen-coated wells were then incubated with dilutions of preimmune or postimmune serum (100 pL) for 1h a t room temperature. Wells were washed with PBS and incubated with peroxidaseconjugated goat anti-rabbit immunoglobulin antibody (1: 2000 in PBS) for 1 h. Following washes with PBS, the wells were incubated with 100 pL of 0.05% (w/v) 2,2'azinobis(3-ethylbenzthiazoline-6-sulfonate~ (ABTS) dissolved in 0.1 M sodium phosphate, 0.08 M citric acid, pH 4.0, and 0.003% (vlv) hydrogen peroxide. Absorbance readings a t 405 nm were recorded with a Titretek Multiscan MCCl340 plate reader. Western Immunoblot Analysis. Following SDSPAGE, the protein bands were electrophoretically transferred to nitrocellulose membranes (Bio-Rad Lab., Hercules, CA) using a Polyblot transfer system (American Bionetics, Hayward, CA). Membranes were then treated for 1h in 2% (wlv) Carnation powdered milk in TBS (100 mM Tris-HC1, 0.15 M NaC1, pH 7.4) followed by a 2 h incubation step with a 1:500 dilution of rabbit anti hEGF (1-51) antisera in TBS containing 0.2% (wlv) BSA or Carnation powdered milk. The membranes were washed and exposed to peroxidase-conjugated goat anti-rabbit immunoglobulin antibody (1:2000 in TBS) for 1 h. The presence of antibody complexes on membranes was detected using the method of Young (25). Briefly, washed membranes were incubated in a solution containing 10
mg of 4-chloro-1-naphthol and 30 mg of 3,3'-diaminobenzidine tetrahydrochloride dissolved in 5 mL of methanol and combined with 40 mL of PBS and 10 pL of 30% (v/v) hydrogen peroxide. Color development was stopped by washing the membranes with distilled water. Radioiodination of hEGF(1-51). Human EGF(151) (2 pg) was labeled to a specific activity of 0.25 pCi/ng (1 mCihmo1) with NalZ5Iusing Chloramine T as previously described (26). Radioligand Binding Assays. MDA-MB-468breast cancer cells (1.5 x lo6 cells) were dispensed into 35 mm culture dishes and incubated for 1h at 37 "C with 1 ng of 1251-hEGF(1-51)in the presence of increasing amounts of unlabeled MCP-4-EDTA-S-MB-hEGF(1-51) or hEGF(1-51) prepared in 0.2% (wlv) human serum albumin in PBS. The cells were then transferred to polystyrene tubes and centrifuged a t 2000 rpm for 5 min to separate the cell pellet and the supernatant. The radioactivity associated with cell pellet and supernatant fractions were then measured in a y scintillation counter. The percentage of lZ5I-labeledhEGF(1-51) bound to breast cancer cells was plotted as a function of the total amount of MCP-4-EDTA-S-MB-hEGF(1-51) or hEGF(1-51) added (Figure 6). lllIn Labeling of the hEGF(1-51) Conjugate. MCP-4-EDTA-S-MB-hEGF(1-51) (18 pg; 2.1 nmol) was dissolved in 0.1 M citrate buffer pH 6.5 and mixed with 3.6 x mol (1.8 mCi) of lllIn chloride, to a specific activity of -50 mCi/mg. The labeling of the conjugate was followed using ITLC-SG plates developed in 0.1 M citrate buffer pH 4.5. In the presence of this mobile phase, the radiolabeled conjugate remained a t the origin and the free IllIn migrated with the solvent front. The flexible chromatographic plate was then cut into 1 2 x 1 cm sections and the radioactivity associated with each segment was measured in a y counter (Figure 5A). Unbound IllIn was removed by purification on a BioGel P-30 column (Pasteur pipette) eluted with 0.1 M citrate buffer pH 4.5. Column fractions of 100 pL were collected (Figure 5B).
Bioconjugate Chem., Vol. 6,No. 6,1995 687
Metal Binding Peptide-EGF Conjugate
A J.U
0
2
6
4
I
..-
1
3
4
5
6
7
8
9 1 0 1 1 1 2
Segment from origin on ITLC sheet [cm]
Column volume [mL]
B
B kDa
2
4
5
6
30.0 -
16.9 10.7 -
6.2 Figure 4. (A) Superimposed elution profiles of hEGF(1-51) (W, MCP-4-EDTA-S-MB-hEGF(1-51) conjugate (O), and of the MCP-4-EDTA-SH peptide ( 0 )on a BioGel P-30 gel filtration column. Absorbance readings for hEGF( 1-51) and its conjugate were recorded a t 405 nm and represent ELISA assay measurements of hEGF(1-51) or its conjugate in column fractions. The presence of MCP-4-EDTA-SH in column fractions was detected at 280 nm. The hatched region highlighted in the elution profile of the conjugate indicates a typical pool of column fractions that would be recovered from this purification step. (B) Coomassie Blue stained Tris-tricine SDS-PAGE gel (lanes 1-3) and corresponding Western immunoblot (lanes 4-6): lane 1, 2 pg of hEGF(1-51); lane 2 , 4 pg of MCP-4-EDTA-S-MB-hEGF(l51) conjugate; lane 3, 10 pg of MCP-4-EDTA-SH peptide; lane 4, 2 pg of hEGF(1-51); lane 5, 4 pg of MCP-4-EDTA-S-MBhEGF(1-51); lane 6, 10 pg of MCP-4-EDTA-SH peptide. All methods are described in the Experimental Procedures. RESULTS AND DISCUSSION
Overall Design of an EGF Construct that Incorporates a Metal-Chelating Peptide. The creation of heterobifunctional peptides that incorporate clusters of metal-chelating sites as well as chemically reactive groups represents a relatively unexplored strategy for producing new radiopharmaceutical agents. In particular, the coupling of such constructs to unique sites on targeting agents such as peptide hormones or antibodies would create well-defined, selective conjugates that could be labeled to potentially higher levels of specific activity. To design and facilitate the assembly of metal-chelating peptides (MCP), we combined the use of peptide branching strategies (27) and protected forms of metal chelators (19) into existing procedures in solid-phase peptide synthesis. In designing the prototypic metal-chelating
0.0
1 .o
2.0
Column volume [mL]
Figure 5. (A)Assessment of radiolabeling efficiency of the conjugate by thin-layer chromatography. An aliquot of the lllInMCP-4-EDTA-S-MB-hEGF(1-51) labeling mixture (4 pL) was spotted and developed on ITLC-SG plates. The radioactivity associated with each 1cm segment on the plate was measured in a y-counter. The labeled conjugate remained a t the origin on the plate (segments 1-2) while unbound indium-111migrated with the solvent front (segment 11).(B) Elution profile of the radiolabeled conjugate on a BioGel P-30 column. The IIIInlabeled conjugate eluted in the void volume of the column. The amount of residual free indium-111 (-15% of total counts) was estimated by counting the column itself following the elution of the radiolabeled conjugate. All methods are described in the Experimental Procedures.
peptide MCP-4-EDTA-SH (Figure l),we introduced a unique thiol moiety a t its C-terminus to couple this MCP to a targeting molecule. For several reasons, the human epidermal growth factor (hEGF) represented a useful and important peptide model for evaluating the impact of introducing MCP-4-EDTA-SH into a targeting agent. Firstly, hEGF is a small, single chain peptide of 53 amino acid (MW 6 kD) which is synthesized in lactating mammary glands, kidney, and submaxillary glands (28,291. The overexpression of its receptor on the surface of breast tumor cells is associated with a poor prognosis in breast cancer patients (4). Secondly, the primary sequence of hEGF does not include lysine residues, and its single N-terminal amino group has been successfully derivatized in the past with fluorescein (30) or rhodamine (31) and ferritin (32) functionalities. Thus, only one MCP-4EDTA-SH can be theoretically incorporated into this conjugate, eliminating an important source of structural
688 Bioconjugate Chem., Vol. 6,No. 6, 1995
heterogeneity often observed in immunoconjugates. Thirdly, a fluoresceinated conjugate of hEGF exhibited similar binding affinities to native EGF (30),suggesting that the introduction of MCP-4-EDTA-SH at the Nterminus of hEGF would not drastically affect the complexation of the construct to the EGF receptor. Finally, the mass of hEGF in relation to MCP-4-EDTA-SHoffered advantages over the use of antibodies, in terms of characterizing and purifying the final construct using standard electrophoretic and chromatographic techniques. Experimentally, a recombinant form of human EGF, abbreviated hEGF(1-51) (33), was used to construct our metal-chelating-hEGF conjugate. This EGF analogue lacks the final two non-essential C-terminus residues of hEGF (34). Design and Synthesis of MCP-CEDTA-SH. The bifunctional metal-chelating-peptide was termed MCP4-EDTA-SH to reflect the presence of four EDTA groups able to bind with high affinity a large spectrum of multivalent cations and the incorporation of a single thiol group located a t its C-terminus that permits its unidirectional coupling to other molecules. As illustrated in Figure 1, the first residue coupled to the solid support was cysteine. The protected thiol group on the side chain of the first residue served as the reactive site for incorporating the peptide construct into hEGF( 1-51). The second residue introduced was aminocaproic acid. It acts as a molecular spacer between the peptide branches and the C-terminus reactive group. It also permits one to monitor the quality of the conjugation step with hEGF(1-51), since this residue is absent in proteins and can be quantified by amino acid analysis. Branching of the peptide was then initiated following the coupling of alanine and tyrosine. Briefly, (Na-Boc, NE-Bocl-lysine, an amino acid having its amino groups a t the Ca and CE positions protected with the same acid labile Boc protecting group, was introduced on the peptide resin. The approach of creating branched peptides using the two amino groups of lysine was popularized by Tam for the construction of immunogenic peptides (27). After the two Boc groups were cleaved with TFA, branching was initiated by coupling 2 equiv of (Na,N+bis-Boc)lysine to the two available amino groups. After another round of acid deprotection, amino caproic acid (ACA)was coupled to the four free amino groups now available. Upon removal of Boc groups, four resulting amino groups were exposed to permit the coupling of glycine residues. Protected EDTA-like groups (19)were finally coupled to the exposed amino groups of glycine residues, and the polymer was detached from the resin support yielding MCP-4-EDTA-SH. The structure of the branched peptide was confirmed by amino acid analysis and mass spectroscopy (see Experimental Procedures). The unidirectional incorporation of MCP-4-EDTA-SH into the maleimide-derivatized hEGF(1-51) results in conjugates that can be easily radiolabeled. Following pathway a outlined in Figure 2, the N-terminus of hEGF(1-51) was reacted with the bifunctional crosslinking agent, (m-maleimidobenzoyl)-N-hydroxysulfosuccinimide ester (sulfo-MBS). The maleimide derivative, abbreviated MB-hEGF(1-51), was desalted on a BioGel P-6 column to remove excess unreacted cross-linker. MBhEGF(1-51) selectively and rapidly reacted with the thiol group of the side chain of the cysteine present in MCP-4-EDTA-SH peptide. The thiol group on MCP-4EDTA-SH was reduced with sodium borohydride (26)and monitored using a DTNB assay before the coupling step with MB-hEGF(1-51) was initiated (Figure 3). The resulting thiol-containing peptide was reacted immediately with MB-hEGF(1-511, and the conjugate was
Remy et al.
desalted on a BioGel P-30 column. The conjugate eluted as a broad band in the volume of the column separated from unreacted MB-hEGF(1-51) and MCP-4-EDTA-SH (Figure 4A). The incorporation of a single branched peptide in the conjugate was confirmed by amino acid analysis (see Experimental Procedures) and SDS polyacrylamide gel electrophoresis. To further confirm that the broad band observed in SDS-PAGE represented the hEGF(1-51) conjugate, we performed western blot analyses on the conjugate using anti-hEGF(1-51) serum. The broad Coomassie Blue stained band observed on SDSPAGE gel comigrated with a positive signal on western blots proving the presence of hEGF(1-51) in the conjugate (Figure 4B). In addition, results from both the Coomassie stained gel and the western blot clearly point out the lack of free hEGF(1-51) contamination in the resulting conjugate preparation. The broadness of the band observed for the conjugate reflects the nature of the peptide MCP-4-EDTA-SH itself. The presence of the branched peptide was not revealed by Coomassie Blue (Figure 4B, lane 3) or silver staining (result not shown). However, when the peptide alone was radioiodinated, autoradiograms of the SDS-PAGE gel show a broad band for the peptide (result not shown). The EDTA groups on the peptide contribute 16 carboxylic arms (with four different pKa‘s) t o the conjugate. Consequently, up to 16 negative charges may be displayed on the branched peptide and its conjugates. Since migration on SDSPAGE is dependent on a proper association of negatively charged SDS molecules with a protein, any heterogeneity in the charge to mass ratio of the conjugate due to the EDTA carboxylic arms would result in a broadening on the corresponding band on the gel. EDTA-like chelators form “stable” chelate complexes with indium-111 (35). This radioisotope with its 3-day half-life and pure y emission is suitable for radioimaging (36). In the labeling step, the hEGF(1-51) conjugate was concentrated and then dissolved in a minimum volume of citrate buffer pH 6.5 before adding “carrier free” 1111nC13. The yield of recovered radioactivity associated with the conjugate after gel filtration was typically > 75% of the starting radioactivity. Analysis of the lllIn labeling reaction mixture by ITLC-SG plates showed the disappearance of the free isotope (indium citrate; Rf1.0) after 30 min (Figure 5A). Most of the activity on the BioGel P-30 column was associated with the void volume peak containing the “‘In-labeled conjugate whereas the free radionuclide is included (Figure 5B). The specific activity of the labeled conjugate was typically between 50 and 60 pCilpg protein. The Conjugate Binds to Epidermal Growth Factor Receptors Expressed on the Breast Cancer Cell Line MDA-MB-468. The binding of hEGF(1-51) conjugate to the EGFRs on MDA-MB-468breast cancer cells was assessed by measuring its ability to compete with 1251-labeledhEGF(1-51) for the receptor sites. As shown conin Figure 6, the MCP-4-EDTA-S-MB-hEGF(1-51) jugate displaced, in a concentration-dependent manner, the binding of radioiodinated hEGF(1-51) to EGFRs with an affinity constant approximately 40-fold lower than hEGF(1-51). This partial loss in receptor affinity may reflect steric hindrance due to the addition of the branched peptide (2.8 kDa) to hEGF(1-51) (5.8 kDa). In summary, we report the design and rapid synthesis of a metal-chelating peptide that incorporates four EDTA groups and one thiol group. The assembly of MCP-4EDTA-SH was facile. The flexibility of the synthetic approach suggests that our initial design can be easily modified to alter the nature of the chelator, the number and composition of the branches, and the coupling
Metal Binding Peptide-EGF Conjugate
Bioconjugate Chem., Vol. 6, No. 6,1995 689
sa
,301
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.I
1
10
100
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Unlabeled peptide added (nM) Figure 6. Displacement curves of 1251-labeled hEGF(1-51) bound to its receptor on breast cancer cells by hEGF(1-51) and its conjugate. Displacement curves of 1251-hEGF(1-51) binding to MDA-MB-468 cells were constructed using increasing concentrations of either hEGF(1-51) (0)or the MCP-4-EDTA-SMB-hEGF(1-51) conjugate (m). All methods are described in the Experimental Procedures.
strategy to targeting agents. Human EGF(1-51) was successfully derivatized with a single MCP-4-EDTA-SH yielding a conjugate that binds specifically to its receptor on breast cancer cells. The conjugate labeled well with indium-111. Experiments are now in progress to assess the localization potential of this radiopharmaceutical agent in nude mice bearing MDA-MB-468 breast tumor xenografts. ACKNOWLEDGMENT
This work was supported by a grant from the National Cancer Institute of Canada, with funds from the Canadian Cancer Society. We thank Dr. Henrianna Pang (University of Toronto) and Lorne Burke (University of Alberta) for performing mass spectrometry measurements, as well as Dr. Arthur Grey of the Nuclear Magnetic Resonance Laboratory, University of Toronto, for useful suggestions. We wish to acknowledge the expertise and technical support of Jim Ferguson. LITERATURE CITED (1) Boring, C. C., Squires, T. S., Tong, T., and Montgomery, S. (1994) Cancer statistics. 1994 C A Cancer J . Clin. 44, 7-26. (2) Gullick, W. J. (1990) Growth factors and oncogenes in breast cancer. Prog. Growth Factor Res. 2, 1-13. (3) Freiss, G., Prebois, C., and Vignon, F. (1993) Control of breast cancer cell growth by steroids and growth factors: interactions and mechanisms. Breast Cancer Res. Treat. 27, 57-68. (4) Stainsbury, J. R. C., Farndon, J. R., Needham, G. K., Malcolm, A. J., and Harris, A. L. (1987) Epidermal growth factor receptor status as predictor of early recurrence of and death from breast cancer. Lancet i , 1398-1402. (5) Baselga, J., and Mendelsohn, J., (1994) The epidermal growth factor receptor as a target for therapy in breast carcinoma. Breast Cancer Res. Treat. 29, 127-138. (6) LeMaistre, F., Meneghetti, C., Howes, L., and Osborne C. K. (1994) Targeting the EGF receptor in breast cancer treatment. Breast Cancer Res. Treat. 32, 97-103. ( 7 ) Merino, M. J., Monteagudo, C., and Neumann, R. D. (1991) Monoclonal antibodies for radioimmunoscintigraphy of breast cancer. Nucl. Med. Biol. 18, 437-443. (8) Kalofonos, H. P., Pawlikowska, T. R., Hemingway, A., and Epenetos, A. A. (1989) Antibody-guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor and placental alkaline phosphatase. J. Nucl. Med. 30, 1636-1645.
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