1Department of Biotechnology, AlbaNova University Center, Kungl Tekniska Högskolan (KTH), SE-106 91 Stockholm, 2Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, 3Affibody AB, PO Box 20137, SE-161 02 Bromma, Sweden and 4Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
5 To whom correspondence should be addressed. E-mail: stefans{at}biotech.kth.se
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: affibody/HER2/ligand/phage display/selection
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although monoclonal antibodies offer many attractive features for tumor targeting, they are also correlated with high manufacturing costs, various side effects and limited efficacy. This might be due to their rather large size, causing slow blood clearance, liver uptake and poor tissue penetration. Therefore, smaller antibody fragments, such as F(ab) (Winter et al., 1994), diabody (Holliger et al., 1993
), scFv (Bird et al., 1988
; Huston et al., 1988
), Fv (Davies and Reichmann, 1996
) and various single domain antibodies (Desmyter et al., 2002
; Holt et al., 2003
) have been developed (Nilsson et al., 2000
).
Antibody fragment approaches are indeed promising, although some limitations seem to exist. Stability issues, both in terms of proteolysis and shelf-life, are obvious challenges, and antibody fragments do not normally fold inside cells, since they contain disulfide bridges, whose formation is crucial for the correct folding of the molecules. The various antibody approaches have recently been complemented by efforts based on different protein scaffolds (Nygren and Uhlén, 1997; Skerra, 2000a
,b
), e.g. trinectins (Koide et al., 2002
; Xu et al., 2002
), anticalins (Skerra, 2000b
) and affibody ligands (Nord et al., 1997
). These engineered protein domains have initially been investigated for various biotechnological applications but different therapeutic approaches might also be envisioned.
A class of affinity ligands denoted affibody molecules has been described earlier (Nord et al., 1997, 2000
; Hansson et al., 1999
; Eklund et al., 2002
; Rönnmark et al., 2002a
). Affibody ligands are based on a 58 amino acid residue protein domain, derived from one of the IgG-binding domains of staphylococcal protein A. This cysteine-free three-helix bundle domain, designated the Z domain (Nilsson et al., 1987
), has been used as a scaffold for construction of combinatorial phagemid libraries from which affibody variants can be selected that target desired molecules using phage display technology. The simple, robust structure of the affibody molecules, together with their low molecular weight (7 kDa), make them suitable for a wide variety of applications. Documented efficacy has been shown in bioprocess- and laboratory-scale bioseparations (Nord et al., 2000
, 2001
; Gräslund et al., 2002
), and promising results have been obtained when evaluating affibody ligands as detection reagents (Karlström and Nygren, 2001
; Rönnmark et al., 2002b
), to engineer adenoviral tropism (Henning et al., 2002
) and to inhibit receptor interactions (Sandström et al., 2003
). Thus, affibody ligands might exhibit therapeutic potential and merit further investigation.
One potential cancer target is HER2/neu (also called ErbB2), a transmembrane protein belonging to the human epidermal growth factor tyrosine kinase receptor family. Increased HER2/neu activity is associated with increased proliferation and decreased apoptotic capacity. HER2/neu is often overexpressed in different cancers, including breast and ovarian (Wang and Hung, 2001), but is expressed only to a small extent or not at all in many normal adult tissues (Natali et al., 1990
; Press et al., 1990
). In patients, overexpression of HER2/neu is associated with short disease-free time and decreased overall survival and it has also been shown that the overexpression is often preserved in metastases (Gancberg et al., 2002
). Together, these factors provide strong arguments for the development of targeting strategies directed against HER2/neu.
In this study, we present the selection and characterization of affibody variants binding to a recombinant extracellular domain of HER2/neu (HER2-ECD), which was used as target during biopanning. The novel selected affibody ligands were characterized for their binding to HER2-ECD in biosensor studies. Furthermore, it was investigated whether the selected affibody molecules bound to the same HER2/neu site as the trastuzumab monoclonal antibody. In addition, the specific binding to native HER2/neu, overexpressed in the tumor cell line SKBR-3, was evaluated.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The amber suppressor Escherichia coli strain RRIM15 (Rüther, 1982
) was used as bacterial host for phage production and cloning procedure. The phagemid vector pAffi1 and the construction of the phagemid library used in this study, a portion (
8.7 x 108) of Zlib2002 (3 x 109 members), will be described elsewhere (E.Gunneriusson, unpublished data). Phagemid inserts of selected clones were subcloned into the expression vector pAY81, containing a T7 promoter (Studier et al., 1990
), a
DNA fragment encoding a hexahistidyl (His6) tag and a multiple cloning site, together with a gene conferring resistance to kanamycin. The E.coli strain BL21(DE3) (Novagen, Madison, WI) was used for protein production from the pAY81 plasmid.
Preparation of phage stocks
Preparation of phage stocks from the library (a portion of Zlib2002) and between selections was performed according to previously described procedures (Nord et al., 1997; Hansson et al., 1999
) using the helper phage M13K07 (New England Biolabs, Beverly, MA). PEG/NaCl precipitation yielded phage titers of about 1013 p.f.u./ml.
Phage selections
A 100 kDa recombinant extracellular domain of HER2/neu (HER2-ECD) comprising 624 amino acids, corresponding to nucleotides 2382109 (Horak et al., 2001), was used as target protein during selections. The protein was biotinylated in vitro using EZ-LinkTMSulfo-NHS-LC-Biotin (Pierce, Rockford, IL). A 30-fold molar excess of biotin was added to HER2-ECD in phosphate-buffered saline (PBS; 10 mM phosphate, 137 mM NaCl, pH 7.2), and the mixture was incubated at room temperature for 1 h followed by extensive dialysis against PBS at 4°C to remove the surplus of biotin. The biotinylated target protein was immobilized on streptavidin-coated paramagnetic beads (Dynabeads® M-280 Streptavidin; Dynal A.S., Oslo, Norway). For each round of selection, beads were washed twice with PBS supplemented with 0.1% Tween-20 (PBST), followed by incubation with biotinylated HER2-ECD in at least 250 µl of PBS, pH 7.2, for 30 min at room temperature under continuous rotation (end over end). For round 1, 40 µg of target protein were incubated with 5 mg of beads, for rounds 2 and 3, 20 µg of target protein were incubated with 2.5 mg of beads, and for round 4, 5 µg of target protein were incubated with 0.63 mg of beads. The beads with the immobilized HER2-ECD were thereafter washed once with PBST. This procedure resulted in an immobilization of
5 µg of the target protein per mg of beads, as determined by SDSPAGE analysis.
The four rounds of biopanning were performed as follows. To avoid unspecific binders, all tubes used in this procedure were pretreated with PBST supplemented with 0.1% gelatin. To further avoid binders against the streptavidin present on the paramagnetic beads, 1 ml of the phage stock supplemented with 0.1% gelatin was for round 1 and 2 preincubated with 0.5 mg of the beads, previously washed twice with PBST. The unbound phage stock was after that subjected to biopanning against the HER2-ECD-coated beads for 2 h at room temperature under continuous rotation. The beads were washed once with PBST in round 1, three times in round 2, six times in round 3 and 12 times in round 4. The bound phages were subsequently eluted with 500 µl of 0.1 M glycineHCl, pH 2.2, for 10 min at room temperature, followed by immediate neutralization with 50 µl of 1 M TrisHCl, pH 8.0. The eluted phages were used to infect log phase RRI
M15 cells for 20 min at 37°C. The infected cell suspensions were spread on TYE agar plates (15 g/l agar, 8 g/l NaCl, 10 g/l tryptone and 6 g/l yeast extract), supplemented with 2% glucose and 100 mg/l ampicillin, followed by overnight incubation at 37°C. The grown colonies were collected by resuspension in tryptic soy broth (TSB, 30 g/l; Merck, Darmstadt, Germany), supplemented with 2% glucose and 100 mg/l ampicillin, and a fraction (100 times excess of cells compared to the phage titer after elution) was used for inoculation, leading to the next generation of phage stock. The selection process was monitored by titrating the phage stocks before selection and after elution. A serial dilution of phages was allowed to infect log phase RRI
M15 cells for 20 min at 37°C, followed by plating on TYE agar plates, supplemented with 2% glucose and 100 mg/l ampicillin, and overnight incubation at 37°C.
DNA sequencing
After four rounds of biopanning, DNA sequencing of phagemid (pAffi1) inserts was performed on 49 randomly picked colonies using specific sequencing primers and Big Dye terminators (Amersham Biosciences, Uppsala, Sweden). The Sanger fragments were analyzed on a DNA sequencer ABI Prism® 3700 Analyzer (Applied Biosystems, Foster City, CA). Subcloned DNA fragments were verified by the same procedure.
DNA constructions
DNA fragments encoding different variants of ZHER2/neu were subcloned into the expression vector pAY81. The fragments were digested from the pAffi1 vector with XhoI (ER0691; Fermentas, Vilnius, Lithuania) and SalI (ER0641; Fermentas), and ligated into the pAY81 vector, previously restricted with the same enzymes and dephosphorylated using calf intestine alkaline phosphatase (CIAP; Fermentas). The cleaved DNA fragments were purified on low-melt agarose gel (Pronadisa, Madrid, Spain) prior to ligation with T4 DNA Ligase (Fermentas). The ligations resulted in expression vectors denoted pAY81-ZHER2/neu:no, encoding the different affibody ligands fused to an N-terminal His6 tag, allowing purification by immobilized metal ion affinity chromatography (IMAC). All plasmid preparations were, after cultivation of transformed E.coli cells overnight, performed using QIAprep Spin Miniprep Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions.
Protein production and purification
Selected affibody variants were expressed as His6-tagged fusion proteins from the pAY81 plasmids in E.coli strain BL21(DE3). Cells were inoculated in 15 ml of TSB medium, containing 50 mg/l kanamycin, and grown in shake flasks overnight at 37°C. Fresh TSB (100 ml), supplemented with 5 g/l yeast extract and 50 mg/l kanamycin, was inoculated with 1 ml of the overnight cultures and the cells were grown at 37°C until A600 nm 0.61.0, when gene expression was induced by addition of isopropyl ß-D-thiogalactoside (IPTG; Apollo Scientific, Derbyshire, UK) to a final concentration of 1 mM. After 34 h of cultivation at 37°C, the cell cultures were harvested by centrifugation (4000 g, 15 min, 4°C). The cell pellets were subsequently resuspended in 25 ml of denaturing buffer (6 M GuaHCl, 47.4 mM Na2HPO4, 2.65 mM NaH2PO4, 10 mM TrisHCl, 0.1 M NaCl, pH 8.0), and disrupted by sonication. ß-Mercaptoethanol was added to the cell suspensions to a final concentration of 10 mM and left with magnetic stirring for 1 h at room temperature. Centrifugation at 15 000 g for 10 min at 4°C resulted in pelleted insoluble material and denatured proteins in the supernatant.
The His6-ZHER2/neu fusion proteins were recovered by IMAC purification on Talon Metal Affinity Resin (BD Biosciences, CA) columns under denaturing conditions. The IMAC columns were pulsed twice with 10 ml of denaturing buffer and 10 ml of elution buffer (8 M urea, 0.1 M NaCl, 29.6 mM HAc, 70.4 mM NaAc, 50 mM NaH2PO4, pH 5.0), and equilibrated with 25 ml of denaturing buffer. Supernatants containing the dissolved His6-ZHER2/neu proteins were diluted with 25 ml of denaturing buffer and filtered (0.45 µm; Sartorius, Göttingen, Germany), before the samples were applied to the columns. After washing with 30 ml of denaturing buffer, the bound proteins were released with elution buffer. Renaturation of the purified fusion proteins was performed by extensive dialysis against PBS (10 mM phosphate, 154 mM NaCl, pH 7.2) at 4°C. Protein concentration was calculated from absorbance measurements at 280 nm, using the appropriate extinction coefficient for each protein, and also determined by amino acid analysis (Aminosyraanalyscentralen, Uppsala, Sweden). The purified proteins were further analyzed by SDSPAGE on Trisglycine 16% homogeneous gels, using a Novex system (Invitrogen, Carlsbad, CA).
Biosensor analyses
A BIAcore® 2000 instrument (Biacore AB, Uppsala, Sweden) was used for real-time biospecific interaction analysis (BIA) between selected affibody molecules and the target protein. HER2-ECD (diluted in 10 mM NaAc, pH 4.5) was immobilized (2200 RU) on the carboxylated dextran layer of one flow-cell surface of a CM5 sensor chip (research grade) (Biacore) by amine coupling, according to the manufacturer's instructions. Another flow-cell surface was activated and deactivated to be used as a reference surface, and human IgG (Amersham Biosciences), HIV-1 gp120 (Protein Sciences Corp., Meriden, CT), and an albumin binding region of streptococcal protein G (BB; Ståhl et al., 1999
) were immobilized on separate flow-cell surfaces on CM5 sensor chips, to serve as negative controls. For all affibody samples, the buffer was changed to HBS-EP (5 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4) by gel filtration using NAPTM-10 columns, according to the manufacturer's protocol (Amersham Biosciences), and the samples were thereafter filtrated (0.45 µm; Millipore, Billerica, MA). Binding analyses were performed at 25°C, and HBS-EP was used as running buffer. In a first experiment, 5 µM of each affibody (diluted in HBS-EP) was injected over all surfaces with a flow rate of 5 µl/min. As a negative control, an unrelated affibody was also injected. In a second experiment, the His6-ZHER2/neu:4 and His6-ZHER2/neu:7 affibody variants were subjected to a kinetic analysis, in which the proteins were injected over a HER2-ECD surface at different concentrations (05 µM, with 0.0098 µM as the lowest concentration for His6-ZHER2/neu:4 and 0.0196 µM for His6-ZHER2/neu:7, diluted in HBS-EP) with a flow rate of 30 µl/min. The dissociation equilibrium constant (KD), the association rate constant (ka), and the dissociation rate constant (kd) were calculated using BIAevaluation 3.2 software (Biacore), assuming a one-to-one binding. For the first two Biacore analyses, the samples were run in duplicates in random order, and after each injection the flow cells were regenerated by the injection of 10 mM HCl. In a third experiment, the His6-ZHER2/neu:4 variant was evaluated for binding competition with the monoclonal antibody trastuzumab. To obtain a reference curve, His6-ZHER2/neu:4 was injected over the HER2-ECD surface at a concentration of 5 µM and the surface was regenerated with 10 mM HCl. The HER2-ECD surface was subsequently saturated by repeated injections with trastuzumab at a concentration of 0.2 µM until no significant additional response was observed. Directly following the trastuzumab injections, the same amount of the His6-ZHER2/neu:4 affibody as in the reference experiment was injected.
Cell culture
Human breast cancer cell line SKBR-3, known to express 2x106 HER2/neu per cell, was purchased from ATCC (American Type Culture Collection, Manassas, VA). The cells were cultured in complemented medium, containing RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and PEST (100 IU/ml penicillin and 100 µg/ml streptomycin), all from Biochrom KG (Berlin, Germany). The cells were cultured at 37°C in humified air containing 5% CO2, and seeded in 3 cm Petri dishes 3 days before the experiment.
Radiolabeling
Labeling precursor, N-succinimidyl-p-(trimethylstannyl)benzoate (SPMB), was prepared according to Orlova et al. (2000), and 5 µg of SPMB were added to 5 MBq of 125I in a 5% solution of acetic acid. To start the reaction, 40 µg of chloramine-T (Sigma, St Louis, MO) in aqueous solution were added. During 5 min, the reaction mixture was agitated, and 80 µg of sodium meta-bisulfate (Aldrich, Steinheim, Germany) in aqueous solution were added to stop the reaction. The radiolabeled precursor was added to 40 µg of His6-ZHER2/neu:4 or His6-ZHER2/neu:7 in 0.07 M borate buffer, pH 9.2. The coupling reaction, in which the labeled precursor is conjugated to amine groups on the protein, was performed at room temperature for 45 min with continuous shaking. Labeled affibody molecules were separated from low molecular weight products using a NAPTM-5 size exclusion column (Amersham Biosciences) equilibrated with PBS. The radiolabeled affibody molecules were then analyzed using BIAcore technology to verify that the labeling procedure had not affected the binding affinity to HER2-ECD.
The monoclonal anti-HER2 antibody trastuzumab, Herceptin® (purchased from Apoteket AB, Sweden) desalted on a PD-10 column (Amersham Biosciences), was radiolabeled with 125I using the chloramine-T method previously described (Sundberg et al., 2003).
Cellular tests
To each dish of 750 000 SKBR-3 cells, 28 ng of labeled His6-ZHER2/neu:4, His6-ZHER2/neu:7 or 470 ng trastuzumab in 1 ml of complemented medium, described above, were added. This amount corresponds to a theoretical 1:1 ratio between the targeting agent and HER2 molecules available on the cells. Three dishes without cells were treated in the same way, in order to determine unspecific and not cell-dependent binding. The resulting radioactivity value was subtracted from all others. To analyze the specificity of cell binding and epitope competition between the targeting agents, three dishes were treated not only with labeled affibody or antibody, but also with a 500-fold excess of unlabeled affibody or antibody. After 3 h of incubation at 37°C, the radioactive medium was removed and the dishes were washed rapidly three times with ice-cold serum-free medium. Cells were trypsinated with 0.5 ml trypsin/EDTA solution (0.25%/0.02% in PBS; Biochrom KG) for 15 min at 37°C. The cells were then resuspended in 1 ml of complemented medium, and 0.5 ml of the cell suspension was used for cell counting and the remaining 1 ml was used for radioactivity measurement in an automated gamma counter.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phage display in vitro selection technology was used to isolate novel affibody ligands, binding to the extracellular domain of HER2/neu (HER2-ECD), from a portion of the combinatorial protein library Zlib2002. The library is based on the 58 residue staphylococcal protein A-derived Z domain, and has been constructed by combinatorial substitution mutagenesis of 13 positions located at the surface responsible for Fc binding activity, essentially as described earlier (Nord et al., 1995). The different members (
3 x 109) of the library are monovalently displayed on protein III of M13 bacteriophage particles using a phagemid vector system (Nord et al., 1995
, 1997
).
Four rounds of phage display selection were performed using biotinylated recombinant HER2-ECD as target protein immobilized on streptavidin-coated paramagnetic beads. To increase the selection stringency, decreasing amounts of target protein together with increasing number of washing steps were employed for each round. DNA sequencing performed on 49 randomly picked colonies from the fourth round of biopanning revealed seven different clones, among which one clone was represented 33 times, indicating significant convergence (Figure 1). Such convergence typically indicates better binding affibody molecules (Nord et al., 1997), but all sequences represented more than once were selected for further characterization, i.e. clones 2, 4, 7 and 8. An alignment of the 13 randomized amino acid residues of the seven unique variants is presented in Figure 1, showing sequence homology in certain positions.
|
DNA fragments encoding four different affibody variants (ZHER2/neu:2, ZHER2/neu:4, ZHER2/neu:7, and ZHER2/neu:8) (Figure 1) were subcloned into the pAY81 vector, resulting in expression vectors encoding, under the control of the T7 promoter, the different affibody molecules fused to an N-terminal His6 peptide tag. The His6-ZHER2/neu fusion proteins were expressed intracellularly in E.coli cells, and thereafter purified on IMAC columns under denaturing conditions. Upon SDSPAGE analysis, the proteins were observed as specific bands of expected molecular weight (8.7 kDa), indicating pure and stable affibody molecules (Figure 2 and data not shown). Interestingly, the His6-ZHER2/neu:7 variant was migrating as a somewhat larger protein than the His6-ZHER2/neu:4 variant (Figure 2), although they differ only in eight amino acid positions (Figure 1). Such deviation in migration has been observed earlier for similar proteins (Gräslund et al., 2000). Estimations from absorbance measurements at 280 nm demonstrated high expression levels (100200 mg/l of cell culture) for all four proteins.
|
Purified affibody ligands were analyzed for HER2-ECD binding by real-time biospecific interaction analysis using a BIAcore biosensor instrument. Prior to sample injection, all affibody proteins were subjected to renaturation and the buffer was changed to the running buffer. The different affibody molecules were subsequently injected over separate sensor chip flow-cell surfaces containing the immobilized target protein HER2-ECD and the control proteins IgG, gp120, and BB, respectively. The His6-ZHER2/neu:2, His6-ZHER2/neu:4, and His6-ZHER2/neu:7 affibody proteins were all shown to bind to HER2-ECD, although the binding of His6-ZHER2/neu:4 and His6-ZHER2/neu:7 (Figure 3A) was considerably stronger than the binding of His6-ZHER2/neu:2 (data not shown). For His6-ZHER2/neu:8 and the unrelated control affibody, no obvious binding to HER2-ECD was observed (data not shown). The reason why the His6-ZHER2/neu:8 affibody molecule did not bind its target HER2-ECD in the BIAcore experiment, although having certain sequence similarities to the binding clones (Figure 1), is not obvious and cannot be fully explained from the present data. However, this clone was the least similar of those characterized, and could potentially bind to a slightly different site that might not be optimally presented on the HER2-ECD immobilized on the biosensor chip. Furthermore, as expected, no significant binding could be seen to the control proteins; IgG (Figure 3A), HIV-1 gp120 (data not shown), and streptococcal protein BB (data not shown). These results suggest that the His6-ZHER2/neu:4 and His6-ZHER2/neu:7 affibody variants bind selectively to the target protein HER2-ECD.
|
To investigate if the His6-ZHER2/neu:4 affibody binds to the same HER2-ECD site as the monoclonal antibody trastuzumab, the affibody was injected over a HER2-ECD flow-cell surface, before and after saturation with trastuzumab (Figure 4). First, as a reference, the His6-ZHER2/neu:4 affibody molecule was, as in Figure 3A, allowed to bind to, and dissociate from, the immobilized extracellular domain of HER2/neu (data not shown). Secondly, the trastuzumab monoclonal antibody was allowed to bind to HER2-ECD (Figure 4A), and a typical association curve was obtained. The HER2-ECD surface was subsequently saturated by repeated injections with trastuzumab until no significant additional response was observed (data not shown). Directly following the trastuzumab injections, the same amount of the His6-ZHER2/neu:4 affibody as in the reference experiment was injected (Figure 4B). Both binding curves of His6-ZHER2/neu:4 had a similar appearance and approximately the same amount of affibody could be bound to the surface irrespective of preceding trastuzumab saturation, indicating that the affibody binds to a different site on the HER2-ECD molecule.
|
The His6-ZHER2/neu:4 and His6-ZHER2/neu:7 affibody proteins were indirectly radiolabeled with 125I by amine coupling. Upon Biacore analysis, to determine if the labeling procedure had affected the binding affinity to HER2-ECD, both affibody variants showed retained affinity (data not shown).
Cellular tests
As presented in Figure 5, the His6-ZHER2/neu:4 affibody showed specific binding to SKBR-3 cells, described to express 2x106 HER2/neu molecules per cell. The binding of radiolabeled His6-ZHER2/neu:4 affibody could be efficiently blocked by the addition of an excess of non-labeled affibody, indicating a specific binding to native HER2/neu (Figure 5A). Furthermore, the His6-ZHER2/neu:4 affibody and the monoclonal antibody trastuzumab was found to bind HER2/neu in a non-competitive manner, since the other ligand when added in excess was unable to block binding (Figure 5A and B). This cellular binding assay might in fact suggest that the presence of an excess of trastuzumab monoclonal antibody would improve binding of the His6-ZHER2/neu:4 affibody molecule to HER2/neu (Figure 5A). This effect could potentially be obtained if the trastuzumab binding stabilized the extracellular domain of HER2/neu, but more experimental evidence would need to be available before stating this. These data corroborate the Biacore results presented in Figure 4, and suggest separate binding sites on HER2/neu for the His6-ZHER2/neu:4 affibody molecule and trastuzumab. The binding of the His6-ZHER2/neu:7 affibody molecule to SKBR-3 cells was below the detection limit (data not shown), probably as a result of the faster dissociation rate for this affibody (Figure 3A and C).
|
Using phage display technology we have selected novel affibody ligands that selectively bind to the human epidermal growth factor receptor 2 (HER2/neu), often overexpressed in breast and ovarian cancers. Seven unique sequences were identified and those represented more than once were subjected to further characterization. As expected the most abundant sequence, represented in 33 out of 49 colonies was found to bind with the best affinity, 50 nM, to the extracellular domain of HER2/neu. The His6-ZHER2/neu:4 affibody was shown to bind to both the recombinant HER2-ECD, as demonstrated in Biacore experiments, and native HER2/neu, overexpressed in the tumor cell line SKBR-3, at a different binding site than the monoclonal antibody trastuzumab. Thus, a HER2/neu binding affibody may have the potential for future use in in vivo diagnostics and therapy of breast, ovarian and other HER2/neu overexpressing tumors. It would in future studies be interesting to evaluate dimerization and affinity maturation strategies (Gunneriusson et al., 1999
; Nord et al., 2001
) to increase the binding strength of the affibody for improved performance. Successful preparation of HER2/neu-binding affibody ligands may have important implications for radionuclide tumor targeting, for in vivo imaging purposes or potentially even radiotherapeutic applications. Since affibody molecules are significantly smaller (20 times) than full-sized monoclonal antibodies, they might prove better in penetration of solid tumors. Comparative studies between antibodies and their fragments have demonstrated that smaller targeting molecules provided faster blood clearance and better tumor penetration (Yokota et al., 1992
). These factors contribute to better imaging contrast in diagnostics (Behr et al., 1995
), and possibly also to better radiotherapeutic efficacy (Ugur et al., 1996
). The half-life of several unrelated affibody molecules seems to be in a rather suitable range for imaging purposes, being in the range of 15 min in mice (data not shown). Radiolabeled receptor ligands, which are even smaller than antibody fragments, also seem to be very promising targeting agents. They typically exhibit rapid clearance from both blood and receptor-negative tissues and have good tumor penetration properties (Froidevaux et al., 2000
). A number of other peptide ligands, are under investigation for clinical use (Heppeler et al., 2000
). Certain tumor-associated antigens, such as HER2/neu, Ep-CAM, CEA and PSMA, are either not receptors or have no identified natural ligands. In these cases where natural ligands cannot be used as low molecular weight targeting agents, affibody ligands may prove to be promising alternatives for tumor targeting. The efficiency of in vivo targeting for imaging purposes, as well as potential therapeutic efficacy, remain to be investigated in future studies.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baselga,J. and Mendelsohn,J. (1994) Pharmacol. Ther., 64, 127154.[CrossRef][ISI][Medline]
Behr,T.M., Becker,W.S., Klein,M.W., Bair,H.J., Scheele,J.R. and Wolf,F.G. (1995) Cancer Res., 55, 5786s5793s.[Abstract]
Bird,R.E., Hardman,K.D., Jacobson,J.W., Johnson,S., Kaufman,B.M., Lee,S.M., Lee,T., Pope,S.H., Riordan,G.S. and Whitlow,M. (1988) Science, 242, 423426.[ISI][Medline]
Davies,J. and Riechmann,L. (1996) Protein Eng., 9, 531537.[ISI][Medline]
Desmyter,A., Spinelli,S., Payan,F., Lauwereys,M., Wyns,L., Muyldermans,S. and Cambillau,C. (2002) J. Biol. Chem., 277, 2364523650.
Eklund,M., Axelsson,L., Uhlén,M. and Nygren,P.-Å. (2002) Proteins, 48, 454462.[CrossRef][ISI][Medline]
Fagerberg,J., Frodin,J.E., Ragnhammar,P., Steinitz,M., Wigzell,H. and Mellstedt,H. (1994) Cancer Immunol. Immunother., 38, 149159.[CrossRef][ISI][Medline]
Froidevaux,S., Heppeler,A., Eberle,A.N., Meier,A.M., Hausler,M., Beglinger,C., Behe,M., Powell,P. and Macke,H.R. (2000) Endocrinology, 141, 33043312.
Gancberg,D., Di Leo,A., Cardoso,F., Rouas,G., Pedrocchi,M., Paesmans,M., Verhest,A., Bernard-Marty,C., Piccart,M.J. and Larsimont,D. (2002) Ann. Oncol., 13, 10361043.
Garnett,M.C., Embleton,M.J., Jacobs,E. and Baldwin,R.W. (1985) Anticancer Drug Des., 1, 312.[Medline]
Gräslund,T., Lundin,G., Uhlén,M., Nygren,P.-Å. and Hober,S. (2000) Protein Eng., 13, 703709.[CrossRef][ISI][Medline]
Gräslund,S., Eklund,M., Falk,R., Uhlén,M., Nygren,P.-Å. and Ståhl,S. (2002) J. Biotechnol., 99, 4150.[CrossRef][ISI][Medline]
Gunneriusson,E., Nord,K., Uhlén,M. and Nygren,P.-Å. (1999) Protein Eng., 12, 873878.[CrossRef][ISI][Medline]
Hansson,M., Ringdahl,J., Robert,A., Power,U., Goetsch,L., Nguyen,T.N., Uhlén,M., Ståhl,S. and Nygren,P.-Å. (1999) Immunotechnology, 4, 237252.[CrossRef][ISI][Medline]
Henning,P., Magnusson,M.K., Gunneriusson,E., Hong,S.S., Boulanger,P., Nygren,P.-Å. and Lindholm,L. (2002) Hum. Gene Ther., 13, 14271439.[CrossRef][ISI][Medline]
Heppeler,A., Froidevaux,S., Eberle,A.N. and Maecke,H.R. (2000) Curr. Med. Chem., 7, 971994.[ISI][Medline]
Holliger,P., Prospero,T. and Winter,G. (1993) Proc. Natl Acad. Sci. USA, 90, 64446448.[Abstract]
Holt,L.J., Herring,C., Jespers,L.S., Woolven,B.P. and Tomlinson,I.M. (2003) Trends Biotechnol., 21, 484490.[CrossRef][ISI][Medline]
Horak,E.M., Heitner,T., Garrison,J.L., Simmons,H.H., Alpaugh,R.K., Amoroso,A.R., Marks,J.D., Weiner,L.M. and Adams,G.P. (2001) Proc. Am. Assoc. Cancer Res., 42, 774.
Huston,J.L., Levinson,D., Mudgett,H.M., Tai,M.S., Novotny,J., Margolies,M.N., Ridge,R.J., Bruccoleri,R.E., Haber,E., Crea,R. and Oppermann,H. (1988) Proc. Natl Acad. Sci. USA, 85, 58795883.[Abstract]
Karlström,A. and Nygren,P.-Å. (2001) Anal. Biochem., 295, 2230.[CrossRef][ISI][Medline]
Koide,A., Abbatiello,S., Rothgery,L. and Koide,S. (2002) Proc. Natl Acad. Sci. USA, 99, 12531258.
Kreitman,R.J. (1999) Curr. Opin. Immunol., 11, 570578.[CrossRef][ISI][Medline]
Natali,P.G., Nicotra,M.R., Bigotti,A., Venturo,I., Slamon,D.J., Fendly,B.M. and Ullrich,A. (1990) Int. J. Cancer, 45, 457461.[ISI][Medline]
Nilsson,B., Moks,T., Jansson,B., Abrahmsen,L., Elmblad,A., Holmgren,E., Henrichson,C., Jones,T.A. and Uhlén,M. (1987) Protein Eng., 1, 107113.[ISI][Medline]
Nilsson,F., Tarli,L., Viti,F. and Neri,D. (2000) Adv. Drug Deliv. Rev., 43, 165196.[CrossRef][ISI][Medline]
Nord,K., Nilsson,J., Nilsson,B., Uhlén,M. and Nygren,P.-Å. (1995) Protein Eng., 8, 601608.[ISI][Medline]
Nord,K., Gunneriusson,E., Ringdahl,J., Ståhl,S., Uhlén,M. and Nygren,P.-Å. (1997) Nat. Biotechnol., 15, 772777.[ISI][Medline]
Nord,K., Gunneriusson,E., Uhlén,M. and Nygren,P.-Å. (2000) J. Biotechnol., 80, 4554.[CrossRef][ISI][Medline]
Nord,K., Nord,O., Uhlén,M., Kelley,B., Ljungqvist,C. and Nygren,P.-Å. (2001) Eur. J. Biochem., 268, 42694277.
Nygren,P.-Å. and Uhlén,M. (1997) Curr. Opin. Struct. Biol., 7, 463469.[CrossRef][ISI][Medline]
Orlova,A., Bruskin,A., Sjöström,A., Lundqvist,H., Gedda,L. and Tolmachev,V. (2000) Nucl. Med. Biol., 27, 827835.[CrossRef][ISI][Medline]
Press,M.F., Cordon-Cardo,C. and Slamon,D.J. (1990) Oncogene, 5, 953962.[ISI][Medline]
Rönnmark,J., Grönlund,H., Uhlén,M. and Nygren,P.-Å. (2002a) Eur. J. Biochem., 269, 26472655.
Rönnmark,J., Hansson,M., Nguyen,T., Uhlén,M., Robert,A., Ståhl,S. and Nygren,P.-Å. (2002b) J. Immunol. Methods, 261, 199211.[CrossRef][ISI][Medline]
Rüther,U. (1982) Nucleic Acids Res., 10, 57655772.[Abstract]
Sandström,K., Xu,Z., Forsberg,G. and Nygren,P.-Å. (2003) Protein Eng., 16, 691697.[CrossRef][ISI][Medline]
Skerra,A. (2000a) J. Mol. Recognit., 13, 167187.[CrossRef][ISI][Medline]
Skerra,A. (2000b) Biochim. Biophys. Acta, 1482, 337350.[ISI][Medline]
Slamon,D.J., Clark,G.M., Wong,S.G., Levin,W.J., Ullrich,A. and McGuire,W.L. (1987) Science, 235, 177182.[ISI][Medline]
Ståhl,S., Nilsson,J., Hober,S., Uhlén,M. and Nygren,P.-Å. (1999) In Flickinger,M.C. and Drew,S.W. (eds), The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation: Affinity Fusions, Gene Expression. John Wiley and Sons Inc., New York, pp. 4963.
Steplewski,Z., Lubeck,M.D. and Koprowski,H. (1983) Science, 221, 865867.[ISI][Medline]
Studier,F.W., Rosenberg,A.H., Dunn,J.J. and Dubendorff,J.W. (1990) Methods Enzymol., 185, 6089.[Medline]
Sundberg,A.L., Blomquist,E., Carlsson,J., Steffen,A.C. and Gedda,L. (2003) Nucl. Med. Biol., 30, 303315.[CrossRef][ISI][Medline]
Trauth,B.C., Klas,C., Peters,A.M., Matzku,S., Moller,P., Falk,W., Debatin,K.M. and Krammer,P.H. (1989) Science, 245, 301305.[ISI][Medline]
Ugur,O., Kostakoglu,L., Hui,E.T., Fisher,D.R., Garmestani,K., Gansow,O.A., Cheung,N.K. and Larson,S.M. (1996) Nucl. Med. Biol., 23, 18.[CrossRef][ISI][Medline]
Wang,S.C. and Hung,M.C. (2001) Semin. Oncol., 28, 115124.[CrossRef][ISI][Medline]
Wilder,R.B., DeNardo,G.L. and DeNardo,S.J. (1996) J. Clin. Oncol., 14, 13831400.[Abstract]
Winter,G., Griffiths,A.D., Hawkins,R.E. and Hoogenboom,H.R. (1994) Annu. Rev. Immunol., 12, 433455.[CrossRef][ISI][Medline]
Xu,L. et al. (2002) Chem. Biol., 9, 933942.[CrossRef][ISI][Medline]
Yokota,T., Milenic,D.E., Whitlow,M. and Schlom,J. (1992) Cancer, 52, 34023408.
Received March 10, 2004; revised May 17, 2004; accepted May 26, 2004.
Edited by Mathias Uhlen