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Generation and Evaluation of Bispecific Affibody Molecules for Simultaneous Targeting of EGFR and HER2 Lina Ekerljung,†,# Helena Wållberg,‡,# Azita Sohrabian,† Karl Andersson,†,§ Mikaela Friedman,∥ Fredrik Y Frejd,†,⊥ Stefan Ståhl,*,‡ and Lars Gedda† †

Department of Radiology, Oncology and Radiation Sciences and ∥Department of Genetics and Pathology, Uppsala University, Sweden ‡ Division of Molecular Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden § Ridgeview Instruments AB, Uppsala, Sweden ⊥ Affibody AB, Stockholm, Sweden S Supporting Information *

ABSTRACT: Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly4Ser)3 or (Ser4Gly)3, and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser4Gly)3 linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly4Ser)3. It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.

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INTRODUCTION

decreased rate of ligand dissociation and receptor downregulation.3 The EGFR family is a common target for both diagnostic and therapeutic applications. Several drugs have been developed with the intention to prevent oncogenic signaling by the receptors. Attempts have been made to interrupt dimerization and thus prevent “oncogenic effects” of the EGFRs. One such drug is the HER2-binding antibody pertuzumab (Omnitarg) which prevents HER2-dimerization.5 Another possible approach is dual targeting of receptors where a binder that simultaneously targets two receptors could potentially prevent activating dimerization by steric interference.6 Simultaneous targeting of two different receptors could also be used to improve the selective delivery of potent payloads to tumor lesions. Tuned bispecific binders could potentially be used to implicitly target receptors that are expressed also in normal tissue. By making a bispecific binder that binds moderately well

The EGFR-family consists of four members; EGFR, HER2, HER3, and HER4 (also known as ErbB1−4). The most extensively studied receptors are EGFR and HER2, while less is known about the other two. Abnormal expression and signaling of the receptors are associated with development and progression of several forms of cancer, and also associated with enhanced invasiveness and resistance to chemotherapy and radiation.1,2 Activation of the receptors occurs through homo- or heterodimerization with another member of the EGFR family, resulting in trans-phosphorylation of tyrosine residues in the intracellular part of the receptor. These phosphorylation sites serve as initiation points for various signaling pathways leading to cellular processes such as proliferation, migration, and apoptosis. The effect on downstream signaling, and hence the biological outcome, depends on the composition of the receptor pair and the identity of the ligand.3 HER2 is considered as the preferred dimerization partner for the other receptors,4 and heterodimerization with HER2 leads to more potent signaling, for example, by © 2012 American Chemical Society

Received: February 9, 2012 Revised: August 7, 2012 Published: August 12, 2012 1802

dx.doi.org/10.1021/bc3000645 | Bioconjugate Chem. 2012, 23, 1802−1811

Bioconjugate Chemistry

Article

Figure 1. (A) Schematic overview of the six bispecific constructs designed and evaluated in this study, including their monomeric parent molecules. The bispecific binders are from now on denoted as constructs I−VI. The theoretical molecular weights are indicated to the right (kDa). (B) SDSPAGE analysis of the IMAC-purified bispecific affibody molecules. Lanes I−VI: bispecific affibody molecules; lane 7, H6-ZHER2:4; lane 8, H6-ZHER2:342; lane 9, H6-ZEGFR:1907; lane M, marker proteins. Molecular weights are indicated in kDa.

gene for Kanamycin (Km) resistance. In addition, pAY430 carries a gene sequence for an N-terminal hexahistidine tag (His6-tag) and a C-terminal cysteine for purification purposes and site-specific labeling, respectively. DNA Constructions. In order to construct affibody molecules with dual specificities, an EGFR-binding affibody molecule, ZEGFR:1907,12 and a HER2-binding affibody molecule, either the first generation binder ZHER2:413 or the second generation binder ZHER2:342,14 displaying affinities toward HER2 of 50 nM and 22 pM, respectively, were recombinantly fused together head-to-tail. The resulting fusion proteins were separated by a 20-amino-acid-long linker sequence comprising either (SSSSG)-repeats or (GGGGS)-repeats for flexibility and separation of the binding domains. Moreover, variants of the bispecific construct where the EGFR- and HER2-binding arms were placed either at the N-terminus or the C-terminus were constructed. A schematic representation of the proteins can be seen in Figure 1A. The six bispecific binders are from now on denoted as constructs I−VI. Each bispecific construct was assembled by amplification of the respective EGFR and HER2 affibody sequences (described in detail by Friedman et al.,12 Wikman et al.,13 and Orlova et al.14) in two separate PCR reactions and subsequent ligation of the resulting overlapping gene fragments. In a first reaction, the gene sequence of the affibody molecule to be placed at the Nterminus was PCR amplified using a forward primer introducing a PstI restriction site and a reverse primer introducing the first part of the linker sequence and a Sf iI restriction site (Supporting Information). In a second reaction, the second part of the linker sequence, with a Sf iI restriction site, was introduced by the forward primer used to PCR amplify the affibody molecule to be placed at the C-terminus in the bispecific construct. Furthermore an AccI restriction site was introduced by the reverse primer used in the second PCR reaction (Supporting Information). In addition, monomeric variants of ZEGFR:1907, ZHER2:4, and ZHER2:342 were constructed as controls by amplification of the corresponding genes using forward and reverse primers with PstI and AccI restriction sites,

to two different receptors, of which one is expressed also in normal tissue, the avidity effect will keep the bispecific binder bound mainly to cells expressing both receptors. The more a receptor is abundantly expressed in normal tissue, the weaker respective binder should be selected for the bispecific construct. This opens up the possibility to target EGFR positive tumors without excessive uptake in EGFR-expressing normal tissues. Coexpression of EGFR and HER2 has been reported in several cancers, and has been found as a prognostic marker in both NSCLC7 and breast cancer.8 This suggests that targeting both EGFR and HER2 may be of prognostic value and potentially advantageous in a treatment approach. A first-generation bispecific affinity protein, generated from dimeric affibody molecules that target EGFR and HER2, has previously been generated and studied.9 In the present study, we have generated bispecific affinity proteins based on monomeric EGFR and HER2 affibody molecules. The rational for this was that the very high affinity obtained by using dimeric affibody molecules, especially for the HER2-binder, would dominate the binding of the bifunctional construct as discussed in Friedman et al.9 In the present study, we also investigate the influence of the linker and the positioning of the affibody molecules. Bispecific constructs were generated containing either of two different linkers, (S4G)3 or (G4S)3, and with the EGFR- and HER2-binding domains at either the N- or the Cterminus. The binding of the ligands, both in Biacore and to living cells, were studied together with their biological effects on receptor dimerization, phosphorylation, cellular growth, and survival after irradiation.

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MATERIALS AND METHODS Bacterial Strains and Vectors. The Escherichia coli (E. coli) bacterial strain RR1ΔM1510 was used as host for all molecular cloning procedures and the E. coli strain BL21(DE3) was used for protein expression purposes. All bispecific constructs were subcloned into the expression vector pAY430, containing a multiple cloning site (the AccI and PstI restriction sites were employed here), a T7 promoter,11 and a 1803



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(Agilent) according to the manufacturer’s recommendations. NEM-conjugated affibody molecules were used in all subsequent biosensor and cell studies. Binding Analysis Using SPR. A Biacore 3000 instrument (GE Healthcare) was used to study the binding interaction between the six bispecific affibody molecules and the two targets EGFR and HER2. Recombinant human EGFR-Fc (hEGFR-Fc) and human HER2-Fc (hHER2-Fc) was immobilized on two of the flow-cell surfaces of a CM-5 sensor chip (GE Healthcare) according to the manufacturer’s instructions. To a third flow-cell, a solution of equal amounts of EGFR-Fc and HER2-Fc was used to immobilize both targets on the same surface. One surface of the chip was activated and deactivated and used as a reference surface in all experiments. In order to verify retained binding to EGFR and HER2 of the respective binding arms, the six NEM-capped, bispecific affibody molecules were diluted to 500 nM and 125 nM in HBS-EP buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.4) and subjected to kinetic analysis of their binding to EGFR and HER2. The proteins were injected over all surfaces at a flow rate of 30 μL/min for 3 min, followed by dissociation for 5 min. Monomeric ZEGFR:1907, ZHER2:4, and ZHER2:342 were injected likewise as controls. All samples were run in duplicate. In a second SPR biosensor experiment, the bispecific affibody molecules were investigated for their ability to bind EGFR and HER2 simultaneously in a sandwich-like setup. The bispecific proteins were diluted to 500 nM in HBS-EP buffer and injected over all surfaces at a flow rate of 30 μL/min. Following an association phase of 6 min, hEGFR-Fc or hHER2-Fc, diluted to a concentration of 100 nM in HBS-EP, was injected for an additional 4 min, followed by dissociation for 5.5 min. After all injection cycles, the flow-cell surfaces were regenerated by injection of 15 μL 10 mM HCl. Data evaluation and estimation of the kinetic parameters ka and kd were performed using the software TraceDrawer 1.1 (Ridgeview Instruments AB) assuming a one-to-one binding model. Cell Lines. The human breast cancer cell line SKBR-3, the gastric carcinoma cell line NCI-N87, and the human squamous carcinoma cell line A-431 were purchased from ATCC (American Type Culture Collection). SKBR-3 cells express approximately 6 × 106 HER2 and 4 × 105 EGFR receptors per cell.15 A-431 expresses 2 × 106 EGFR and 105 HER2.15 We have not been able to detect HER2 by Western blot in A-431 cells without prior immunoprecipitation. NCI-N87 express relatively high levels of both receptors.16 SKBR-3 and NCI-N87 cells were cultivated in RPMI1640, and A-431 cells in Ham's F10. All cell culture media were supplemented with 10% fetal serum albumin (FBS), 2 mM L-glutamin, 100 IU/mL penicillin, and 100 μg/mL streptomycin. LigandTracer Binding Assay. 20 MBq of 125I was added to 5 μL of N-succinimidyl p-(trimethylstannyl)benzoate (SPMB) (1 mg/mL 5% acetic acid in methanol). Radiolabeling was initiated by adding 20 μL of chloramine-T (2 mg/ mL, 5% acetic acid in MeOH) and mixed for 5 min. Labeling reaction was terminated by adding 40 μL sodium metabisulfite (NBS) (2 mg/mL in water). Reaction mixture evaporated to dryness with argon gas followed by addition of 40−60 μg of the bispecific affibody molecules (1 μg/μL in 0.1 M borate buffer pH 9). The coupling reactions continued for 1.5 h with continuous shaking. Reaction mixtures were separated on NAP5 columns and the high-molecular-weight fractions containing

respectively (Supporting Information). All PCR products were purified using a QIAquick PCR purification kit (QIAGEN) before digestion of the DNA fragments and the expression vector pAY430 (described above) with the appropriate restriction enzymes and subsequent ligation of inserts and vector. The resulting plasmids, encoding six different bispecific EGFR × HER2 constructs and three monomeric controls with an N-terminal His6-tag and a C-terminal cysteine, were verified by sequencing before transformation into BL21(DE3) cells by heat chock for protein expression. Protein Expression and Purification. The nine sequence confirmed affibody variants were expressed from pAY430 as His6-tagged fusion proteins in the E. coli strain BL21(DE3). Transformed cells were grown overnight (ON) in 10 mL TSB + Y medium (30 g/L tryptic soy broth, 5 g/L yeast extract) supplemented with 50 μg/mL Km at 37 °C, 150 rpm. One milliliter of the ON culture was used for inoculation of 100 mL fresh medium (TSB + Y, 50 μg/mL Km) in shake flasks. The cells were grown at 37 °C, 200 rpm until 0.5 < OD600 < 1, upon which gene expression was induced by addition of isopropyl-Lthio-β-D-galactopyranoside (IPTG) to a final concentration of 1 mM and protein expression was continued at 25 °C, 150 rpm ON. The cells were harvested by centrifugation, 2600 × g, 4 °C for 8 min, and the cell pellets stored at −20 °C until protein purification. The nine His6-tagged affibody variants were purified by immobilized metal ion chromatography (IMAC) under denaturing conditions. The cell pellets were dissolved in lysis buffer (7 M guanidine HCl, 47 mM Na2HPO4, 2.65 mM NaH2PO4, 10 mM Tris−HCl, 100 mM NaCl, and 20 mM βmercaptoethanol, pH 8.0) and the cells lysed for 2 h at 37 °C under shaking before centrifugation, 30 000 × g for 20 min. The supernatants were passed through a 45 μm filter before loading on equilibrated (lysis buffer) HisPur Cobalt Resin (Thermo Fisher Scientific Inc., Rochester, NY, USA) columns. The bound proteins were washed with lysis buffer before elution with elution buffer (6 M urea, 50 mM NaH2PO4, 100 mM NaCl, 30 mM acetic acid, and 70 mM Na-acetate, pH 5.0) and subsequent buffer exchange of the purified proteins to PBS (2.68 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, and 8.1 mM Na2HPO4, pH 7.4) using PD-10 columns (GE Healthcare) according to the manufacturer’s recommendations. The purity and identity of the recovered proteins were assessed by analyzing the proteins on SDS-PAGE, followed by Coomassie staining. Finally, in order to determine the concentration of the purified proteins, the absorbance at 280 nm was measured. Conjugation with N-Ethylmaleimide. In order to prevent potential dimer formation due to the C-terminal cysteines and consequent interference with future experiments, the cysteines of the six bispecific and the three monomeric affibody molecules were capped by conjugation with N-ethylmaleimide (NEM). Approximately 2 mg of purified protein in 50 mM Tris-HCl, pH 8, was reduced with 30 mM dithiothreitol (DTT) for 30 min at 40 °C under shaking. Excess DTT was removed by passing the protein through a NAP-5 size exclusion chromatography (SEC) column (GE Healthcare) and the buffer was exchanged to 0.1 M NaAc, pH 6. A 5-fold excess of NEM was added to the reduced proteins and incubated at 40 °C for 1 h, after which unreacted NEM was removed by again passing the proteins over a SEC column and the buffer was exchanged to PBS. The efficiency of the conjugation with NEM was evaluated using mass spectrometry on an Agilent Technologies 6520 Accurate-Mass Q-TOF LC/MS system 1804



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Table 1. Kinetic Data from the SPR Biosensor Analysis of the Bispecific Affibody Molecules EGFRa ka (M I II III IV V VI a b

1.1 1.7 6.8 1.1 1.3 1.5

−1

× × × × × ×

−1 b

s ) 104 104 103 104 104 104

−1 b

kd (s ) 5.3 5.0 5.9 6.1 5.6 5.2

× × × × × ×

10−4 10−4 10−4 10−4 10−4 10−4

EGFR × HER2a

HER2a KD (nM) 47 29 86 55 43 35

b

ka (M 9.6 9.8 5.3 4.2 1.1 8.7

−1

× × × × × ×

−1 b

s ) 104 104 104 104 105 104

−1 b

kd (s ) 1.1 1.7 3.2 4.1 1.3 1.9

× × × × × ×

10−2 10−2 10−4 10−4 10−2 10−2

KD (nM) 115 172 6 10 118 219

b

ka (M

−1

−1 b

s )

ND ND 4.7 × 104 3.9 × 104 ND ND

kd (s−1)b

KD (nM)b

ND ND 1.7 × 10−4 1.3 × 10−5 ND ND

ND ND 4 0.3 ND ND

The analytes, bispecific affibody molecules, were injected over immobilized EGFR, HER2, and a mixed surface containing both EGFR and HER2. Kinetic parameters were estimated using TraceDrawer. cND = not determined

the 125I-labeled proteins were eluted in 1 mL according to the manufacturer’s recommendations. SKBR-3 or A-431 cells were seeded on a local part of a cell dish (as described by Björke et al.17) and cultivated until the cell number was approximately 106. The 125I-labeled bispecific affibody molecule was added to the cell dish and real-time ligand−cell interaction was monitored in room temperature with LigandTracer Grey (Ridgeview Instruments AB) by measurement of 125I.18,19 The concentration of ligand ranged from 20 to 210 nM depending on the calculated affinity for the receptors (data from SPR biosensor analysis, Table 1). Three experimental set-ups on both cell lines were used for all six binders: uptake at three different concentrations followed by retention, and two control experiments where one of the receptors was blocked by preincubation with 10-fold molar excess of a monovalent nonlabeled affibody molecule, ZEGFR:1907, ZHER2:4, or ZHER2:342. Data evaluation and estimation of the equilibrium dissociation constant KD were performed using the software TraceDrawer 1.3 (Ridgeview Instruments AB) using a one-to-one binding model. Receptor Phosphorylation and Dimerization Assays. Cell lysis and Western blotting was performed as previously described.20 Antibodies specific for HER2 and phosphotyrosine (pY99) were from Santa Cruz Biotechnology. The anti-EGFR antibody was from Cell Signaling Technology. Antimouse and antirabbit antibodies linked with horseradish peroxidase were purchased from Invitrogen. The antibody directed to β-actin was from Sigma-Aldrich. All antibodies were used according to the manufacturer’s instructions in PBS-T with 1% BSA and 0.1% NaAzide. For the dimerization study, the cells were washed with PBS and then incubated with 5 mM BS3 (Bis(Sulfosuccinimidyl) suberate) (Thermo Fisher Scientific Inc.) for 30 min at room temperature. To end the cross-linking reaction, 1 M Tris-Cl was added to a final concentration of 20 mM and set on bench for 15 min. After this, the cells were lysed according to standard protocols and subjected to SDS-PAGE on NuPAGE 3−8% Tris-Acetate gels. DuoSet IC kit measuring phospho-EGFR and phospho-HER2 by ELISA were purchased from R&D Systems. Clonogenic Assay. Subconfluent cultures of SKBR-3 cells were treated with 10 nM ZHER2:342, construct IV, or left untreated for 2 h at 37 °C. Thereafter, the cells were irradiated with 137Cs γ-ray photons at a dose rate of 1.034 Gy/min. The total dose was 4 or 8 Gy. Then, the cells were allowed to repair for about 16 h at 37 °C before trypsinized and reseeded at suitable concentrations. The cells were cultured for 3 weeks and then fixed in 97% ethanol and stained with hematoxylin. Colonies with more than 50 cells were counted. Proliferation Assay. SKBR-3 and NCI-N87 cells were treated with 20 nM construct II or left untreated. The culture

media and substances were replaced twice a week. Before the cells became confluent, they were trypsinized and counted. After this, a subpopulation was reseeded for further cultivation. The growth curves are calculated as if all cells were reseeded at each subcultivation. Three parallel culture flasks were kept for each experiment. The cell growth was monitored for 35 days in total. Statistical Methods. Unpaired t tests were used to test for significant differences both in phosphorylation levels and survival. If P < 0.05, the difference was considered as significant.

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RESULTS Generation of Bispecific EGFR × HER2 Affibody Molecules. The six bispecific affibody molecules ZHER2:4(S4G)3-ZEGFR:1907, ZEGFR:1907-(S4G)3-ZHER2:4, ZHER2:342-(S4G)3Z EGFR:1907 , Z EGFR:1907 -(S 4 G) 3 -Z HER2:342 , Z HER2:4 -(G 4 S) 3 ZEGFR:1907, and ZEGFR:1907-(G4S)3-ZHER2:4, hereafter named constructs I−VI (Figure 1A), and the three corresponding monomeric control proteins ZHER2:4, ZHER2:342, and ZEGFR:1907 were expressed in E. coli as His6-tagged fusion proteins. The His6-tag was employed for purification of the affibody molecules by IMAC. Subsequent analysis of the purified proteins by SDS-PAGE revealed pure protein products of the approximate expected molecular weights (Figure 1B). The theoretical molecular weights of all proteins are indicated in Figure 1A. The slight differences in migration pattern are most likely due to the differences in amino acids recruited at the binding surface, since such variations have been observed previously. From Abs280 nm measurements, the concentrations of all purified proteins could be determined, and the average yield was calculated to 120−170 mg purified protein per liter cultivation. All proteins were produced with a C-terminal cysteine to provide means for site-specific modifications of the proteins (e.g., labeling with fluorophores or radioactive isotopes) should that be desired. However, for the initial characterization of the proteins by SPR biosensor analysis, capping of the free cysteines in order to prevent potential dimer formation of the proteins was advisible. Accordingly, the six bispecific affibody molecules and the monomeric control proteins were conjugated with N-ethylmaleimide. Subsequent MS analysis of the conjugated proteins revealed pure products with the correct molecular weight (data not shown). Bispecific EGFR × HER2 Affibody Molecules Show Binding in SPR Analysis. In order to verify preserved functionality of the respective binding arms, the six bispecific affibody molecules were subjected to kinetic binding analysis to EGFR and HER2, using real-time biosensor analysis on a Biacore instrument. The bispecific proteins were injected at different concentrations over surfaces containing immobilized 1805



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Figure 2. Sandwich analysis of the simultaneous binding to EGFR and HER2 measured in Biacore. Soluble EGFR or HER2 was injected after the bispecific affibody molecules III (Black) or IV (Gray) were allowed to bind to immobilized HER2 (A) or EGFR (B). In a second step, the bispecific affibody molecules could capture soluble EGFR or HER2 when already bound to their corresponding immobilized binding partner. Arrows indicate the start and end time points when substances were injected. To the right are schematic representations of the respective experimental setup in (A) and (B).

obtained sensorgrams, it was clear that, when allowed to interact with both receptors on the mixed target surface, all constructs displayed avidity effects and an increase in affinity in an additive manner, strongly suggesting that both binding arms of the bispecific constructs were simultaneously involved in the binding interaction. For constructs III and IV, containing the high-affinity anti-HER2 domain ZHER2:342, the kinetic parameters and affinity for binding to the mixed surface could be determined. The affinity was shown to increase when both binding domains were recruited as compared to when binding to only one of the receptors with one arm (Table 1). In a separate experiment, the simultaneous binding to both targets was investigated by a sandwich assay in which soluble EGFR and HER2 was injected after the bispecific affibody constructs III and IV, which in a first step had been allowed to bind to immobilized EGFR or HER2. The obtained sensorgrams showed that both bispecific variants could capture soluble EGFR and HER2 when already bound to their corresponding immobilized target (Figure 2). For construct IV, simultaneous binding to both targets was possible irrespective of whether the bispecific molecule was allowed to bind EGFR or HER2 first. For construct III, simultaneous binding to both targets was clearly possible when construct III was allowed to first interact with immobilized EGFR and thereafter capture HER2 in solution (Figure 2B). When first allowed to bind immobilized HER2, a weak increase in response when soluble EGFR was injected indicated that

EGFR and HER2, respectively. Sensorgrams obtained from the biosensor analysis showed that both binding arms had preserved functionality and could bind to EGFR and HER2, respectively. Furthermore, it was shown that all bispecific affibody variants could bind to both EGFR and HER2 irrespective of the orientation of the binding domains (N- or C-terminus). Using the binding-curve evaluation software TraceDrawer, the kinetic parameters, ka and kd as well as the dissociation equilibrium constant (KD), for the EGFR and HER2 binding domains of the bispecific molecules interacting with their respective targets could be determined and are presented in Table 1. Since the constructs are bispecific the model used, assuming a 1:1 binding, is not ideal but will result in kinetic values of reasonable accuracy. All constructs displayed a slightly reduced affinity to their respective targets as compared to their monomeric parental molecules (ZHER2:4, KD = 50 nM and ZHER2:342, KD = 22 pM for HER2, and ZEGFR:1907 KD = 5 nM for EGFR).12−14 The reduced affinity could largely be explained by a slower on-rate of the bispecific molecules. It was furthermore observed that the affinity to the target was increased when the binding domain was placed at the N-terminus. This observation was seen for both the EGFR- and HER2-binding affibody domains. To a third surface of the Biacore chip, a mixture of EGFR and HER2 was immobilized and the simultaneous binding of the constructs to both targets was investigated. From the 1806



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Figure 3. Binding curves of 125I-labeled construct I to SKBR-3 cells measured in real time in LigandTracer. (A,B,C) 125I-I was added at 120 nM to a cell dish which had already been incubated with 1.2 μM of unlabeled monomer ligand, ZEGFR:1907 (B), ZHER2:4 (C), or both (A). (A) After preblocking with both ligands, no binding could be monitored. (B,C) After preblocking with one of the ligands, some binding to the cells could be detected, most visible during the association phase. (D) 125I-I was added to the cell dish in three concentrations, 30 nM, 60 nM, and 120 nM. After 7.5 h, the ligand was removed and the retention was followed. After 24 h, some binding could still be detected. Dotted line indicates the reference area were there were no cells, i.e., the background.

simultaneous binding was possible also in this situation, although less pronounced compared to construct IV. However, since both constructs could interact with both targets simultaneously in Biacore, binding studies using living cells were undertaken to further explore the interactions. LigandTracer Cell Binding Assay. The interaction of the bispecific affibody molecules with living cells was analyzed by measuring the real-time binding of 125I-labeled affibody constructs in LigandTracer. SKBR-3 cells, which express both EGFR and HER2 at relatively high levels, were used to investigate if the bispecific affibody ligands could bind both receptors. The second cell line, A-431, mainly express EGFR at high levels. All six binders were tested on both cell lines and for three experimental set-ups: cell binding at three different concentrations followed by retention, and two control experiments in which one of the receptors was blocked by preincubation of 10-fold molar excess of a monovalent nonlabeled affibody molecule, ZEGFR:1907, ZHER2:4, or ZHER2:342. Preblocking of both receptors by adding both ZEGFR:1907 and ZHER2:4 was performed for construct I. Representative binding curves on SKBR-3 cells for 125I-I are shown as an example in Figure 3. Data for all binders are summarized in Table 2 as estimated KD calculated with TraceDrawer using a 1:1-binding

Table 2. Kinetic Data from the Binding Assay in Living Cells on LigandTracer binding to SKBR-3 cells KDa (nM) binding to A-431 cells KDa (nM) no block I II III IV V VI

14 24 5 2 370 1000

2:342

preblock ZEGFR:1907

no block

preblock ZHER2:4 /ZHER2:342

preblock ZEGFR:1907

W W N N N N

W W 6.3 2 W N

7 8 64 0.3 120 28

9 16 N 2.7 W 17

N N N N N N

preblock ZHER2:4/ZHER

a

Kinetic parameters were estimated using TraceDrawer using a 1:1binding model. W = weak binding, no kinetic parameters could be calculated. N = not detectable (no signal of binding was obtained).

model, or indicated as W when KD could not be calculated due to weak binding (signal), or N when no binding was detected. As can be seen in Figure 3A, blocking both receptors suppressed binding of the bispecific affibody molecules completely, while blocking only one of the receptors resulted in weak but visible binding, notable during the association phase (Figure 3B,C). However, without any preblocking, a clear binding rate could be seen during both binding and retention 1807



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Figure 4. Dimerization assay of the bispecific binders. Monomeric affibody molecules and EGF were used as controls. After stimulation by 100 nM of each substance for 1 h at 37 °C, the chemical cross-linker BS3 was added. EGF-treatment was only 5 min to minimize internalization. After cell lysis, the samples were subjected to SDS-PAGE, followed by transfer and Western blot. (A) SKBR-3 cells. (B) A-431 cells.

Figure 5. (A,B) Phosphorylation study of the EGFR receptor by ELISA. SKBR-3 cells (A) or A-431 cells (B) were stimulated by 100 nM of each substance for 1 h at 37 °C. EGF treatment was only 5 min to minimize internalization. To enable comparison between different experiments, the obtained values were normalized to its respective untreated control. The graphs show mean values from 4 experiments with standard deviations. Constructs I−VI and EGF significantly deviate from the untreated control in SKBR-3 cells, while the difference is significant only for EGF in A-431 cells (unpaired t test, P < 0.05).

Receptor Phosphorylation and Dimerization. The effect of the bispecific affibody molecules on receptor phosphorylation and receptor dimerization were measured using Western blot and ELISA. As can be seen in Figure 4A, treatment by the bispecific constructs increased the levels of dimerized EGFR in SKBR-3 cells. The low level of dimerization shown after EGF treatment could be explained by rapid internalization and degradation of EGF-EGFR complexes. It should also be noticed that the assay cannot distinguish between homodimers and heterodimers. The levels of phosphorylated EGFR were also increased in SKBR3 cells when treated with constructs I−VI (Figure 5A). The control affibody molecules in monomeric forms, ZEGFR:1907, ZHER2:4, and ZHER2:342, did not induce any dimerization or phosphorylation differing from the untreated cells. The phosphorylation results were confirmed by Western blot (data not shown). The levels of dimerized and phosphorylated HER2 were already high in unstimulated SKBR-3 cells. This can be explained by the high number of HER2 receptors, since overexpression is believed to lead to spontaneous dimerization and activation.21,22 The six bispecific affibody molecules and EGF all had a tendency to increase the HER2 dimerization and phosphorylation in SKBR3 cells, but due to the high background level, this was not significant (Supporting Information Figure S1). In A-431 cells, none of the affibody molecules changed the receptor dimerization or phosphorylation (Figures 4B and 5B). The level of EGFR dimers was low in unstimulated A-431, but

phases (Figure 3D), indicating an avidity effect of construct I. Similar results were obtained also for construct II. On the other hand, neither of the constructs containing the affinity matured HER2-binder ZHER2:342 (constructs III and IV) showed the same avidity effect on SKBR-3 cells as the constructs with ZHER2:4. Binding to a cell surface where only HER2 was available (i.e., preblocking by ZEGFR:1907 on SKBR-3 cells) resulted in the same apparent affinity as when EGFR was also available (Table 2). Interestingly, the two constructs with the linker (G4S)3 (V and VI) demonstrated lower affinity than the corresponding constructs with the linker (S4G)3 (I and II). For V and VI, a difference in affinity depending on the orientation of ZHER2:4 could also be seen, verifying the observation from the Biacore analysis that positioning at the N-terminus improves affinity. Construct V displayed higher affinity than VI to SKBR-3 cells (Table 2). In the A-431 cell line, no binding could be seen when EGFR was blocked by ZEGFR:1907, indicating that HER2 expression is too low to allow detection in LigandTracer. Constructs II−V showed improved binding to the cells when both receptors were present compared to when HER2 was blocked. It should be noted that, for construct IV, the KD for A-431 is the same as when both receptors were present in the Biacore assay (0.3 nM). Both constructs IV and VI, which have higher affinity for the cells than III and V, respectively, shows that the affinity was favored by positioning of ZEGFR:1907 at the N-terminus. 1808



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Figure 6. Proliferation assay. SKBR-3 and NCI-N87 cells were continuously treated with 20 nM of construct II or left untreated as a control. The cells were counted once a week, and a subpopulation was reseeded for further cultivation. No differences in growth rate were seen.

could be increased by treatment with EGF for 5 min (Figure 4B). Stimulation with EGF also resulted in increased phosphorylation levels of both EGFR (Figure 5B) and HER2; albeit, the level of HER2 was very low (data not shown). Cell Proliferation Assay. To analyze if the bispecific affibody molecules could influence the proliferation of EGFRand HER2-expressing cells, SKBR-3 and NCI-N87 cells were continuously treated with construct II. This binder was selected as the most interesting candidate of the six, since it showed avidity on SKBR-3 cells and that this orientation of the binding domains was preferable compared to construct I. As can be seen in Figure 6, construct II did not affect cell growth compared to untreated cells either in SKBR-3 or NCI-N87 cells. Radiosensitizing Effects Analyzed by a Clonogenic Survival Assay. In order to further assess the biological effect of the bispecific affibody molecules and also compare with the previous bispecific binder from Friedman et al.,9 (ZHER2:342)2(G4S)3-(ZEGFR:1907)2, containing bivalent HER2- and EGFRbinding affibody molecules, a clonogenic survival assay on SKBR-3 cells was set up. Cells were analyzed after treatment with affibody molecules and ionizing radiation. Survival is shown in Figure 7. The four substances could be divided into two groups. Construct IV and monomeric ZHER2:342, which both contain only one HER2-binding affibody moiety, did not differ from the untreated control. Interestingly, a dimeric HER2 binding construct (ZHER2:342)2 and the previously studied bispecific (ZHER2:342)2-(G4S)3-(ZEGFR:1907)2 (denoted as X in Figure 7) significantly decreased survival (P < 0.01). Apparently, the valency of the HER2-binder is of importance for the effect of the ligand. Survival after 4 Gy of γ-irradiation did not differ between the two groups (data not shown).

Figure 7. Clonogenic survival of SKBR-3 cells after irradiation by 8 Gy. Cells were treated with construct IV, ZHER2:342, (ZHER2:342)2(G4S)3-(ZEGFR:1907)2 (denoted as X), or (ZHER2:342)2 for 2 h prior to irradiation and allowed to repair for 16 h before reseeding. After 3 weeks of cultivation, colonies containing more than 50 cells were counted and the survival fraction could be calculated. (ZHER2:342)2 and the bispecific (ZHER2:342)2-(G4S)3-(ZEGFR:1907)2 significantly decreased survival (P < 0.01, unpaired t test), while treatment with ZHER2:342 or construct IV did not differ from the irradiated control. Mean values and standard deviations are calculated on at least 3 replicates.

ZHER2:4, binding to HER2 with an affinity of 50 nM,13 and a second generation, affinity matured binder, ZHER2:342, with an affinity to HER2 of 22 pM.14 The two binding domains, directed at EGFR and HER2, were assembled head-to-tail, separated by a spacer sequence encoding a 20-amino-acid-long glycine- or serine-based linker, to facilitate flexibility and separation of the two binding arms. Furthermore, the influence of the N- or C-terminal positioning of the affibody molecules comprising the bispecific constructs was investigated. Real-time biosensor analyses using Biacore showed that all bispecific constructs retained functionality in both binding arms and, furthermore, were capable of simultaneous interaction with

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DISCUSSION Six different bispecific affibody molecules with specificity to EGFR and HER2 were constructed by assembling gene sequences of previously described individual affibody molecules targeting EGFR or HER2.12−14,23 The EGFR-binding affibody variant, ZEGFR:1907, reported to bind to EGFR with an apparent affinity of 5 nM12 was combined with two different variants of a HER2-binding affibody molecule, a first generation binder 1809



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measured can be explained by the high background level of activated HER2 together with the known differences in receptor number of EGFR and HER2. The small increase of heterodimeric HER2 is probably masked by the large amount of HER2 dimers already existing and therefore cannot be quantified by our methods. Dimerization of the EGFR family is known to be entirely receptor-mediated.6 EGF simultaneously binds to both domain I and III on EGFR and is thereby believed to stabilize the receptor in its “open” configuration where the dimerization loop on domain II is exposed.24 Whether the bispecific affibody molecules can induce functional receptor dimerization could be answered by the ability of the monomeric ZEGFR:1907 to expose the dimerization arm. The HER2-binding part is of less importance since HER2 has its dimerization arm constitutively exposed. A previous study by Nordberg et al.25 shows that (ZEGFR:955)2 partly competes for the same epitope as EGF, and partly with the same epitope as Cetuximab (domain III). ZEGFR:1907 is an affinity matured version of ZEGFR:955 and should therefore bind the same epitope. Hence, ZEGFR:1907 is likely to bind to domain III of EGFR. To evaluate and compare the biological effect of the bispecific affibody molecules in this study, with the previous one described by Friedman et al.,9 we analyzed the ability of the affibody molecules to sensitize for ionizing radiation in SKBR-3 cells. The HER2-binding part, ZHER2:342 and (ZHER2:342)2, of the constructs were used as control substances. Survival after 8 Gy γ-irradiation divided the substances into two groups; construct IV had no effect on the survival, similar to that of its HER2binding part ZHER2:342, while (ZHER2:342)2 and (ZHER2:342)2(G4S)3-(ZEGFR:1907)2 (denoted as X in Figure 7) significantly decreased survival compared to the irradiated control. Apparently, the valency of the binder in the bispecific construct is of importance, as speculated in our previous study.9 Perhaps the “tetravalent” affinity protein (Z HER2:342 ) 2 -(G 4 S) 3 (ZEGFR:1907)2 can bind two EGFR and two HER2 receptors, at least to some extent. Most likely, the receptor binding of (ZHER2:342)2-(G4S)3-(ZEGFR:1907)2 is dominated by the highaffinity ligand (ZHER2:342)2. Furthermore, the proliferation assay showed that construct II had no effect on SKBR-3 or NCI-N87 cell growth. To conclude, we have shown that using monomeric EGFR and HER2 for assembly of bispecific binders, rather than its dimeric equivalents, is more successful for making a functional bispecific affinity protein. Further, we have shown that all three monovalent binders are more functional when positioned at the N-terminal part of the construct and also that the linker (S4G)3 renders higher affinity of the bispecific binders compared to (G4S)3 on living cells. In this study, all six binders can simultaneously bind EGFR and HER2, without large effects on cells. Additional future studies should be conducted in vivo to further investigate the therapeutic effects of the bispecific constructs. This could furthermore advance our understanding of the effects from simultaneous targeting of EGFR and HER2.

both targets. Although the constructs displayed a reduced affinity to the targets compared to the monospecific control proteins, this was largely counteracted when the bispecific molecules were allowed to interact with both target proteins. Constructs III and especially IV showed increased affinity when binding to both EGFR and HER2, compared to when only one receptor was present. The Biacore data furthermore revealed a significant influence from the positioning of the binding domains. An N-terminal positioning was more favorable for the affinity to the respective targets. Also, LigandTracer analysis in living cells supported the observation that the affinity for each target was increased when the binder is placed at the Nterminus. This was most noticeable for constructs V and VI, which were the constructs with the overall lowest affinity. In SKBR-3 cells where the binding predominantly originates from the HER2-binding part, construct V had higher affinity than VI. In A-431 cells where binding is predominantly to EGFR, construct VI had higher affinity than V (Table 2). Interestingly, bispecific constructs containing the novel (S4G)3 linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (G4S)3. Ligand binding to SKBR-3 cells showed that the constructs containing ZHER2:4 and the linker (S4G)3 (I and II) displayed an avidity effect when both receptors were present (Figure 3 and Table 2), indicating that both binding arms of the bispecific constructs were involved in the binding interaction. In A-431 cells, constructs II−V showed increased binding when both receptors were present compared to when only EGFR was available. When EGFR was blocked, no binding could be detected in this cell line. The differences in results between the binding data from the Biacore and the LigandTracer experiments (as well as between the two cell lines) can to a great extent be explained by the big differences in receptor numbers. In the Biacore setup, the EGFR and the HER2 receptor were present at approximately equal numbers, while in LigandTracer, it depended on the cell line. SKBR-3 cells have approximately 6 × 106 HER2 receptors but only 4 × 105 EGFR receptors.15 Furthermore, in Biacore the binding is measured to recombinant proteins in an artificial environment, while the binding in LigandTracer is measured to functional receptors expressed on living cells. The balance in receptor number as well as the difference in affinity for the two targets seems to have large impact on the binding characteristics of a bispecific binder. This can be exemplified by construct IV: In SKBR-3 cells, binding is predominantly originating from the high affinity HER2-binder ZHER2:342 and no apparent contribution from the EGFR-binding part can be seen. In sharp contrast, in A-431 cells and to the mixed receptor surface in the Biacore, the construct shows avidity. The Western blots after cross-linking with BS3 clearly shows that all six bispecific affibody molecules induce EGFR dimerization when added to SKBR-3 cells. However, the assay cannot distinguish between fully functional receptor dimers and receptors that are merely present at the cell membrane in such proximity of each other that they can be cross-linked by BS3. The phosphorylation results, which show a small significant increase in phosphorylated EGFR after treatment with bispecific binders, indicate that the EGFR is functionally dimerized. This is probably a heterodimer with HER2 since the monomeric ZEGFR:1907 does not induce dimers or receptor phosphorylation (Figures 4 and 5). Why no significant HER2 dimerization and phosphorylation could be

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ASSOCIATED CONTENT

S Supporting Information *

List of the sequences of all primers used for the construction of the bispeciffic affibody molecules. Figure S1 shows the Western blot analysis of phosphorylated EGFR in two different cell lines (SKBR-3 and A-431) after treatment with the bispecific affibody molecules. This material is available free of charge via the Internet at http://pubs.acs.org. 1810



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AUTHOR INFORMATION

Corresponding Author

*Phone: +46 8 5537 8329. Fax: +46 8 5537 8481. E-mail: [email protected]. Author Contributions #

Authors contributed equally.

Notes

The authors declare the following competing financial interest(s): One of the authors of this paper, Dr. Fredrik Y. Frejd, has an affiliation (employment) with Affibody AB. Affibody AB holds intellectual property rights and trademarks for affibody molecules. One of the authors of this paper, Dr. Karl Andersson, has an affiliation (employment and shareholder) with Ridgeview Instruments AB. Ridgeview Instruments AB holds intellectual property rights and trademarks for LigandTracer instruments and TraceDrawer software.

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ACKNOWLEDGMENTS This work was supported by grants from the Governmental Agency for Innovation Systems (VINNOVA), the Swedish Cancer Society (Cancerfonden), and the Swedish Research Council (Vetenskapsrådet). The authors are grateful for the technical assistance of Dr. Sebastian Grimm.

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