Scholarly article on topic 'Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with 188Re'

Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with 188Re Academic research paper on "Chemical sciences"

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{Rhenium-188 / Affibody / HER2 / "Peptide-based chelator" / Biodistribution / "Radionuclide therapy"}

Abstract of research paper on Chemical sciences, author of scientific article — Mohamed Altai, Hadis Honarvar, Helena Wållberg, Joanna Strand, Zohreh Varasteh, et al.

Abstract Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

Academic research paper on topic "Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with 188Re"

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European Journal of Medicinal Chemistry

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Original article

Selection of an optimal cysteine-containing peptide-based chelator for

labeling of affibody molecules with 188Re

Mohamed Altai a, Hadis Honarvar a, Helena Wallberg b'c, Joanna Strand a,

Zohreh Varasteh d, Maria Rosestedt d, Anna Orlova d, Finn Dunas e, Mattias Sandstrom f,

John Lofblom b, Vladimir Tolmachev a' *, Stefan Stahl b

a Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden b IKTH Royal Institute of Technology, School of Biotechnology, Division of Protein Technology, Stockholm, Sweden c Division of Biochemistry, Dept. of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden d Preclinical PET Platform, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden e Affibody AB, Stockholm, Sweden

f Medical Physics Uppsala University Hospital, Uppsala, Sweden



Article history:

Received 1 April 2014

Received in revised form

23 September 2014

Accepted 25 September 2014

Available online 26 September 2014

Keywords: Rhenium-188 Affibody HER2

Peptide-based chelator Biodistribution Radionuclide therapy


Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

© 2014 Published by Elsevier Masson SAS.

1. Introduction

The use of monoclonal antibodies against cancer-associated antigens improves survival of patients with disseminated cancer. However, a substantial fraction of tumors show primary drug resistance. Development of a secondary (acquired) resistance has also been identified during the course of targeted therapy despite preserved antigen expression [1—5]. Targeted radionuclide therapy is a promising method for treatment of both inoperable primary and disseminated antibody-resistant tumors. Several classes of radiolabeled proteins have been used for delivery of radionuclides

* Corresponding author. Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.

E-mail address: (V. Tolmachev). 0223-5234/© 2014 Published by Elsevier Masson SAS.

to tumors in vivo, utilizing the specific binding of these agents to phenotypic molecular alterations in malignant cells. In fact, tumor targeting with radiolabeled antibodies for detection of malignancy or for therapeutic purposes has been reported as early as the 1950's [6,7]. However, both preclinical and clinical investigations have revealed that the clinical effect of radioimmunotherapy (RIT) on bulky solid tumors is limited [8]. Due to their large size (150 kDa), radiolabeled intact antibodies are characterized by slow clearance from blood and poor tumor penetration [8]. Several alternative methods have been developed to overcome problems associated with conventional RIT. The use of smaller enzymatically produced antibody fragments [8,9], analogues of naturally existing peptide ligands to receptors overexpressed in tumors [10], and pretargeting strategies [11] constitute examples of new approaches aiming to deliver radionuclides to tumors while sparing healthy tissues. Another promising approach for the development of high affinity

small-size agents for targeted radionuclide therapy is the use of engineered scaffold proteins.

Affibody molecules were one of the first evaluated classes of non-immunoglobulin scaffold proteins for in vivo radionuclide targeting. These high affinity ligands are based on a 58 amino acid (7 kDa) triple a-helical scaffold derived from domain B of staphylococcal protein A [12,13]. Thirteen surface located amino acids on helices 1 and 2 have been randomized, generating large combinatorial libraries from which affibody variants targeting the desired antigen could be selected. Due to their generally high affinity towards specific molecular targets and their small size, affibody molecules are attractive candidates for tumor-targeting applications [14]. An important feature is the robustness of this targeting protein. Affibody molecules typically tolerate the denaturation during labeling procedures (e.g. exposure to high temperature or lipophilic solvents), since they rapidly and completely refold to their initial structure after returning to physiological conditions [15]. This characteristic permits labeling in a pH range from 3.6 [16] to 11.0 [17] and at temperatures up to 90 °C [18], without losing binding capacity. This allows labeling with a large variety of nu-clides and enables to select an optimal one for each particular application. An anti-Human Epidermal Growth Factor Receptor type 2 (HER2) affibody molecule has been extensively investigated, primarily for tumor imaging application. This affibody molecule binds with picomolar affinity (22 pM) to the extracellular domain of HER2 receptors [19].

Cellular processing studies have shown that internalization of anti-HER2 affibody molecules is relatively slow [20]. Typically, less than 30% of the bound tracer was internalized after 24 h. Therefore, the use of a residualizing radiometal label to increase radioactivity retention in tumor cells is of less importance for the anti-HER2 affibody molecule, because most of the retained activity is membrane bound. However, similar to other small proteins and pep-tides, efficient tumor targeting was associated with significant renal accumulation of affibody molecules labeled with residualiz-ing radiometals such as 177Lu, 90Y, and 114mIn [21-23]. This accumulation is attributed to extensive tubular reabsorption after glomerular filtration [24].

A high level of reabsorption exposes the kidneys to irradiation at high doses, which would potentially compromise their functionality. An approach to reduce this high renal dose is fusing affibody molecules to a 46 amino acid albumin binding domain (ABD), derived from streptococcal protein G. The non-covalent binding of such affibody-ABD fusion proteins to albumin in vivo was found to prolong circulation times and reduce kidney accumulation [21,25]. However, long residence time of the conjugate in circulation may result in higher irradiation of the very radiosensitive bone marrow. On the contrary, affibody molecules labeled with non-residualizing labels provide rapid clearance of radioactivity from kidneys without reducing tumor dose while avoiding long circulation times [26].

Earlier, a series of studies involving labeling of affibody molecules with technetium-99m (99mTc) using peptide-based N3S che-lators has been performed (for reviews concerning labeling with technetium and rhenium radioisotopes, see Refs. [27-29]). These studies demonstrated that targeting properties of labeled probes can be improved by selecting an optimal chelator [30-36]. Substitution of the amino acids forming the chelator for binding 99mTc had a profound effect on the biodistribution profile of affibody molecules. Initially, the 99mTc-labeling of affibody molecules was performed using the mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) chelator coupled to the N-terminus of the anti-HER2 affibody molecule ZHER2:342 [37]. The tracer was capable of targeting HER2 in vivo, but underwent high hepatobiliary excretion. Combination of serine (polar) and glutamic acid (charged) in mercaptoacetyl-

containing chelators (maESE) provided a radiolabeled conjugate with less hepatobiliary excretion and renal retention of radioactivity [38]. To permit recombinant production, cysteine as the thiol-bearing moiety was introduced on the C-terminus of the anti-HER2 affibody molecule. The tracer 99mTc-ZHER2:2395-Cys (having KVDC sequence at the C-terminus) demonstrated excellent biodistribution except from an elevated kidney uptake [34]. To reduce the renal uptake, a GSEC chelator (a "mirror" C-terminal homolog of maESE-) was designed [33]. The use of GSEC resulted in four-fold reduction of renal retention in comparison with KVDC. For further optimization of cysteine containing peptide-based chela-tors, a series of recombinantly produced derivatives containing GSEC, GGGC, GGSC, GGEC, or GGKC peptide chelators at the C-ter-minus (designated as Zher2:V1, Zher2:V2, Zher2:V3, Zher2:V4 and ZHER2:V5 respectively) was generated [35]. The in vivo evaluation of the 99mTc-labeled variants demonstrated low hepatobiliary excretion and rapid blood clearance. The renal retention varied considerably, with the GGGC containing anti-HER2 affibody molecule showing the lowest kidney associated radioactivity (6.4 ± 0.6% ID/g at 4 h p.i.). Further optimization showed that the order of amino acids in the chelating sequence influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points [36]. The results obtained with 99mTc-labeled anti-HER2 affibody molecules prompted us to utilize this experience in developing an affibody-based tumor-targeting agent for peptide-based radionuclide therapy.

Rhenium is a chemical analogue of technetium, and two rhenium isotopes, 186Re (T/2 = 3.72 days; Ejmax = 1.08 MeV) and 188Re (T,/2 = 17.0 h; Ej max = 2.1 MeV), are considered suitable for therapy. High-energy (188Re) or medium energy (186Re) b-particles are well suited for eradication of both bulky non-operable tumors and large metastases. The g-radiation of these nuclides (186Re: Eg = 155 keV,15%; 188Re: Eg = 137 keV, 9%) enables in vivo imaging of the biodistribution of rhenium-labeled conjugates, which could be used for patient-specific dosimetry. At the same time, the g-radiation would not contribute too much to the whole-body irradiation due to low abundance. The half-life of 188Re matches better the rapid in vivo kinetics of affibody molecules. In addition, 188Re is produced using a 188W/188Re generator, which reduces the radio-nuclide costs and facilitates the logistics of therapy. Furthermore, this nuclide might be produced in a no-carrier-added state, with a high specific radioactivity. Thus 188Re is an attractive label for affibody-based radionuclide therapy on the precondition that the issue of renal accumulation is solved. Rhenium isotopes (mainly 188Re) have been used for RIT to eliminate large burdens of leukemia in conjunction with standard preparative regimens prior to stem cells transplantation [39,40]. Attempts to expand the therapeutic applications of rhenium, in order to include solid tumors, had limited success. However, 188Re has been preclinically evaluated as a label for, e.g., somatostatin analogues for treatment of neuroendocrine tumors [41], a-melanocyte stimulating hormone peptide analogs (a-MSH) for treatment of melanoma [42], water soluble oligonucleotides (c-MORF) [43] and peptide-based haptens for treatment of colorectal cancer using a pretargeting approach

Earlier, our group has reported 186Re-maGSG-ZHER2:342 as a potential affibody conjugate for therapy of HER2 expressing tumors

[45]. This conjugate had a low renal retention, but rather high degree of hepatobiliary excretion. Dosimetry evaluation showed that dose to tumor should exceed dose to normal tissues by several folds. However, a high irradiation of the intestines was a major concern and prompted us to further optimize the targeting properties and biodistribution profile of the anti-HER2 affibody molecule for therapeutic application using rhenium isotopes.

In this study we evaluate seven affibody molecules containing the amino acid sequences GGGC (ZHer2:V2). GGSC (ZHer2:V3). GGEC (Zher2:V4), GGKC (Zher2:V5), GSGC (Zher2:V6), GEGC (Zher2:V7), and GKGC (ZHER2:V8), as peptide-based chelators (Fig. 1) at the C-ter-minus for labeling with 188Re. Evaluation included binding specificity to HER2 receptors, affinity determination, stability of the conjugates and biodistribution profile of the conjugates in normal and tumor bearing mice. The parental anti-HER2 affibody molecule ZHER2:23g5 containing a KVDC peptide-based chelator at the C-ter-minus was used as a reference.

2. Experimental section

2.1. Materials

D-gluconic acid sodium salt, ethylenediaminetetraacetic acid (EDTA) and chloroquine disphosphate were from Sigma—Aldrich, tin(ll)-chloride dihydrate (SnCl2 x 2H2O) and pyridine were from Fluka Chemika. Phosphate buffered saline, pH 7.4 (PBS) was produced in house.188Re was obtained as perrhenate by elution of a 188W/188Re generator with 4 mL sterile 0.9% sodium chloride (both from Polatom, Otwok-Poland). An automated g-counter with a ~7.6-cm (3-in) Nal(Tl) detector (1480 WIZARD; Wallac Oy) was used to measure radioactivity. A Cyclone storage phosphor system and OptiQuant image analysis software (both from Perkin—Elmer) were used to measure the radioactivity on instant thin-layer chromatography silica gel-impregnated (ITLC SG) strips. The affibody molecule ZHER2:2395 was provided and purified by Affibody AB. The variants ZHER2:V2 to ZHER2:V8 were produced and purified using a novel antiidiotypic purification strategy, which has been developed earlier by Wällberg et al. [46]. Purity of all variants was above 95%. A description of some relevant properties of these variants is presented elsewhere [35,36]. Data on cellular uptake and biodistribution were assessed by an unpaired, two-tailed Student's t-test using GraphPad Prism (v 4.00 for Windows GraphPad Software,

San Diego, California, USA) in order to determine any significant differences (p < 0.05).

2.2. Labeling of affibody molecules with 188Re

The cysteine-containing HER2-binding affibody molecules were site-specifically labeled with 188Re using freeze-dried labeling kits each containing 5 mg of sodium gluconate, 100 mg of edetate disodium (EDTA), and 1 mg of tin(II) chloride dihydrate. The freeze-dried kits were stored at -20 ° C.

Radiolabeling was performed by adding the contents of one freeze-dried kit, dissolved in 1.25 M sodium acetate, pH 4.2,100 mg of each affibody molecule up to a total volume of 100 mL. To the reaction solution, 35 mL (3-4 MBq) of a 188Re-containing generator eluate was added under argon gas. An equivalent of 220 mg ascorbic acid (2 mg/mL in 1.25 M sodium acetate buffer, pH 4.2) was added to the reaction vial and the mixture was incubated at 90 °C for 60 min and then cooled at room temperature for 5 min. Thereafter, the total amount of ascorbic acid in the reaction vial was adjusted to be 1 mg using a solution of 5 mg/mL ascorbic acid in PBS containing 2% BSA. One mL samples were taken for analysis of the labeling yield by ITLC SG with PBS as the mobile phase. The labeled conjugates were purified using disposable NAP-5 columns (GE Healthcare) pre-equilibrated with PBS, containing 2% BSA. The column was eluted with 900 mL of the same solution. The final solution was further diluted with 100 mL PBS containing 2% BSA and 500 mg ascorbic acid to a final volume of 1 mL. After purification, the purity was evaluated using instant thin-layer chromatography.

2.3. In vitro studies

2.3.1. Binding specificity of radiolabeled affibody molecules to HER2-expressing cells in vitro

Binding specificity of eight 188Re-labeled variants was evaluated using ovarian carcinoma SKOV-3 (1.6 x 106 receptors/cell) purchased from American Type Tissue Culture Collection (ATCC). Cells

Fig. 1. Overview of the 188Re-labeled affibody molecule variants evaluated in this study. A. The studied HER2-binding affibody molecules including the C-terminal chelating sequence aligned to the parent molecule Zher2:342. The variable amino acids are marked in bold. Positions of a-helices 1 through 3 are indicated by boxes. B. General structure of the N3S chelators formed by a C-terminal cysteine and three adjacent amino acids. R1 and R2 and denote the side chains of the amino acids involved in the coordination of the radionuclide.

were maintained in Roswell Park Memorial Institute (RPMI) medium (Sigma—Aldrich) containing 10% fetal bovine serum. Briefly, a 2 nM solution of each variant was added to six Petri dishes (~1 x 106 cells/dish). For blocking, a 500-fold excess of non-labeled recombinant ZHER2:2395 was added 5 min before the labeled conjugates to saturate the receptors. The cells were incubated during 1 h in a humidified incubator at 37 °C. Thereafter, the media was collected, the cells were detached by trypsin-EDTA solution and the radioactivity in cells and media was measured to calculate the percentage of cell-bound radioactivity.

2.3.2. Affinity determination using LigandTracer

SKOV-3 cells were seeded on a local area of a cell culture dish (NunclonTM, Size 100620, NUNC A/S, Roskilde, Denmark), as described previously [47]. The binding of 188Re-labeled anti-HER2 affibody molecules to living cells was monitored in real-time using LigandTracer Yellow, using established methods [47]. To suppress internalization of affibody-HER2 complex, measurements were performed at 4 °C. By using the TraceDrawer software, which allows the calculation of both association and dissociation rate, it becomes possible to determine the affinity of radiolabeled conjugate [47]. In order to cover the concentration span needed for proper affinity estimation, two concentrations of 49 pM and 98 pM (selected based on previous KD values obtained using Biacore) of each variant were added in each affinity assay.

2.3.3. Cellular retention of mRe —labeled affibody molecules by HER2 expressing ovarian carcinoma (SKOV-3) cells

Cellular retention was studied using the methods validated for affibody molecules earlier [20]. SKOV-3 cells (1 x 106 cells/dish) were incubated with 2 nM solution of labeled affibody at 4 °C. After 1 h incubation, the medium with the labeled compound was removed and cells were washed three times with ice-cold serumfree medium. One mL of complete media was added to each dish and cells were further incubated at 37 °C in an atmosphere containing 5% CO2. At designated time points (4 h and 24 h), a group of three dishes was removed from the incubator, the media was collected and cells were washed three times with ice-cold serumfree medium. Thereafter, cells were detached by trypsin-EDTA solution and the radioactivity in cells and media was measured to calculate the percentage of cell-bound radioactivity from total added.

2.4. In vivo studies

The animal experiments were planned and performed in accordance with national legislation on laboratory animal protection. The animal study plans have been approved by the local Ethics Committee for Animal Research in Uppsala.

Biodistribution studies were performed in female immuno-competent Naval Medical Research Institute (NMRI) mice. All mice were acclimatized for 1 week at the Rudbeck Laboratory animal facility before any experimental procedures. Sixty four mice with an average weight of 30 g were randomized into groups of four animals each. Animals were injected intravenously with 1 pg (120 kBq) conjugate per animal in 100 pL PBS containing 2% BSA. One group of mice was euthanized at each predetermined time point (1 and 4 h after injection) by an intraperitoneal injection of anesthesia, Ketalar—Rompun solution (20 pL/g body weight; Ketalar: 10 mg/mL, Pfizer; Rompun[xylazine]: 1 mg/mL) with subsequent exsanguination by heart puncture using a 1 mL syringe prewashed with diluted heparin (5000 IU/mL). Blood, lung, liver, spleen, stomach, kidney, salivary glands, muscle, bone, intestines, and the remaining carcass were collected. Organs and tissue samples were weighed, and their radioactivity was measured. The

tissue uptake values were calculated as percent of injected dose per gram tissue (%ID/g) except for the intestines and the carcass, which were calculated as %ID per whole sample.

In vivo imaging was performed to obtain a visual confirmation of the biodistribution data. Three NMRI mice were injected with 0.7 MBq (8 pg) of 188Re-ZHER2:2395 (-KVDC), 188Re-ZHER2:V4 (-GGEC) and 188Re-ZHER2:V2 (-GGGC) respectively. Mice were sacrificed by cervical dislocation at 4 h after injection. The imaging experiment was performed using an Infinia g-camera (GE Healthcare) equipped with a High-energy general purpose (HEGP) collimator. Static images (30 min) were obtained with a zoom factor of2ina256 x 256 matrix.

The 188Re-labeled conjugates with the lowest and highest uptake in normal organs, Re-ZHER2:V2 (-GGGC) and 188Re-ZHER2 :2395 (-KVDC), respectively, was used in a tumor targeting study with the aim of determining tumor and kidney accumulation of radioactivity. A total of eight Balb/C nude mice (average weight 19 g) were subcutaneously injected with 1 x 107 HER2 -expressing SKOV-3 cells in the left flank. Three weeks after implantation the tumors were 3 mm [3] in size and mice were randomized in to two groups (n = 4). Each group was injected intravenously with either 188Re-ZHER2:V2 (-GGGC) or 188Re-ZHER2:2395 (-KVDC) (total protein dose 5 pg per animal, about 90 kBq in 100 pL PBS containing 2% BSA). Mice were sacrificed at 4 h p.i. and previously mentioned organs in addition to the tumors were collected, weighed, and their radioactivity was measured. For specificity control, one additional group (n = 4) was pre-injected with 500 pg of ZHER2:342 45 min before injection of 188Re-ZHER2:V2 to saturate HER2 receptors in tumors. The animals were sacrificed at 4 h p.i., and uptake in blood and tumors was measured.

3. Results

3.1. Labeling

All affibody molecules were efficiently labeled with 188Re by transchelation from gluconate.

Data concerning the labeling yield and radiochemical purity of Zher2:2395 and the — GXYC variants; Zher2:V2 to Zher2:V8, are presented in Table 1. The labeling yields exceeded 95% for all variants except ZHER2:2395 for which the labeling yield was 89.3 ± 1.2%. The radiochemical purity of the labeled products after purification using NAP-5 columns was above 99% for all the eight conjugates.

3.2. Binding to HER2-expressing cells in vitro

All conjugates showed specific binding to the HER2 receptor as the uptake by SKOV-3 cells was significantly (p-value < 5 x 10~4) reduced by pre-saturation with 500-fold molar excess of unlabeled protein.

Table 1

Radiochemical yield and radiochemical purity of different anti-HER2 affibody molecules.

Variant Yield% Purity%

188Re-ZHER2:2395 89.3 ± 1.2 99.9

lssRe-ZHER2:V2 95.8 ± 1 100

188Re-ZHER2:V3 96.6 ± 0.6 100

188Re-ZHER2:V6 97 ± 0.8 100

188Re-ZHER2:V4 98.2 ± 0.7 100

188Re-ZHER2:V7 95.8 ± 0.1 100

188Re-ZHER2:V5 96.5 ± 0.2 100

188Re-ZHER2:V8 96.5 ± 0.1 100

The kinetics measurements of all the eight 188Re-labeled affi-body molecules binding to HER2-expressing SKOV-3 cells in vitro using LigandTracer confirmed that high affinity to HER2 was preserved after labeling. Evaluation of data by fitting the binding and dissociation curves to a 1:1 interaction model indicates that the binding of radiolabeled conjugates to living HER2-expressing cells is mediated by single binding site populations. This 1:1 interaction is in agreement with previously obtained Surface Plasmon Resonance (SPR) analysis data performed on a Biacore 2000 instrument (GE Healthcare) [36]. The dissociation constants at equilibrium (KD) for binding of radiolabeled affibody molecules to the HER2 receptor are shown in Table 2. All variants demonstrated high affinity in the low picomolar range. Fig. 2 shows the KD measurement sensogram

for 188Re-ZHER2:V2.

The composition of the peptide-based chelators had an apparent influence on the cellular retention of the 188Re-labeled conjugates after binding to HER2-expressing SKOV-3 cells (Fig. 3).188Re-ZHER2:2395 and 188Re-ZHER2:V5 (both containing the positively charged amino acid lysine) showed the highest overall retention compared to the other variants, 188Re-ZHER2:V3 (-GGSC) and 188Re-ZHER2 :V4 (-GGEC) containing a polar and a negatively charged amino acid, respectively, showed moderate overall retention after 24 h. 188Re-ZHER2:V2 (-GGGC) with no polar or charged residues had the lowest overall retention both at 4 and 24 h. Moreover, Zher2:V6 (-GSGC), Zher2:V7 (-GEGC) and Zher2:V8 (-GKGC) showed an appreciable difference from the levels of cellular retention obtained for their counterparts with GGXC chelators (Zher2:V3, Zher2:V4 and Zher2:V5). In general, the GXGC-containing conjugates were less efficiently retained by HER2 expressing cells.

3.3. In vivo studies

To determine the in vivo uptake of radioactivity in different organs, the radiolabeled constructs were injected into female NMRI mice. Data concerning biodistribution 1 and 4 h p.i, are presented in Table 3. For comparison, data of the GXGC and GGXC-containing counterparts are provided in the form of pairs. All comparative findings reported are statistically significant unless otherwise stated. All eight studied 188Re-labeled constructs, ZhER2:2395,


showed a rapid blood clearance, which is typical for affibody molecules. The blood-associated radioactivity was reduced 5—8.2-fold between 1 and 4 h after injection. A low level of radioactivity in the gastrointestinal tract (with its content) suggested that hep-atobiliary excretion did not play a substantial role. No significant differences in radioactivity were observed in organs that rapidly establish an equilibrium with the blood pool (spleen and salivary

Table 2

The dissociation constants KD of interaction of rhenium-

188 labeled affibody molecules with HER2-expressing

SKOV-3 cells determined using LigandTracer at 4 °C.

Affinity determination using ligand tracer

Variant Kd (pM)

1SSRe-ZHER2:2395 10. 6 ± 0.6

1SSRe-ZHER2:V2 6.4 ± 0.4

Re-ZHER2:V3 51 ±3

Re-ZHER2:V6 4 ± 1

1SSRe-ZHER2:V4 10 ±6

1S S Re-ZHER2:V7 21 ± 10

Re-ZHER2:V5 5± 2

Re-ZHER2:VS <13 pMa

a Accurate measurement of off-rate was not possible

despite 6 h long measurement due to very slow dissociation.

glands). All other non-excretory organs displayed similar uptake profiles of the different affibody variants.

Overall, the analysis demonstrated a strong influence of the composition of the peptide-based chelator and the relative positions of amino acids on the biodistribution. Most obvious was the influence on uptake in liver, lung and kidneys (Fig. 4). It is apparent that the lysine containing variants 188Re-ZHER2:2395 (KVDC), 188Re-ZhER2:V5 (GGKC)and188Re-ZHER2 :V8 (GKGC) were retained to a higher extent in the excretory organs in comparison to other variants. Moreover, changing the position of a single amino acid on the peptide based chelator from GGXC to GXGC resulted in a significantly lower retention in the liver (1.44 ± 0.06 %ID/g for ZHER2:V3 vs. 0.7 ± 0.1%ID/g for ZHER2:V6,1.3 ± 0.3 %ID/g for ZHER2:V4 vs. 0.8 ± 0.1 % ID/g for ZHER2:V7 and 3.3 ± 0.5%ID/g for ZHER2:V5 vs. 2.4 ± 0.1 %ID/g for ZHER2:V8) and kidneys (20 ± 4 %ID/g for ZHER2:V3 vs. 10 ± 1 %ID/g for Zher2:V6, 31 ± 6 %ID/g for Zher2:V4 vs. 23 ± 2 %ID/g for Zher2:V7 and 152 ± 24 %ID/g for ZHER2:V5 vs. 136 ± 23 %ID/g for ZHER2:V8) at 4 h after injection. A somewhat elevated lung uptake of 188Re-ZHER2:V4 was observed both at 1 and 4 h after injection (3.2 ± 0.8 and 1.0 ± 0.3 %ID/ g respectively), compared to the other variants. This elevated pulmonary uptake has been observed earlier for 99mTc-labeled ZHER2:V4 at 4 h p.i. [35]. Throughout the study the variant, 188Re-ZHER2:V2, with the C-terminal chelating sequence —GGGC, provided the lowest radioactivity retention in all organs including the kidneys (3.1 ± 0.5

%ID/g 4 h p.i.) and liver (0.33 ± 0.05 %ID/g 4 h p.i.).

188 188

A comparative imaging study using Re-ZHER2:2395, Re-

ZHER2:V4 and 188Re-ZHER2 :V2 respectively confirmed the biodistribution results (Fig. 5). Images obtained at 4 h after injection showed no visible uptake in any organ except from kidneys. The intensity of kidney visualization varied between 188Re-ZHER2:2395 (strong) and 188Re-ZHER2:V4 (moderate). The kidneys were barely visible when mice were injected with 188Re-ZHER2:V2.

To evaluate influence of chelators on tumor targeting, we performed a comparative biodistribution study between 188Re-ZHER2:V2 and 188Re-ZHER2:2395 in female NMRI nu/nu mice bearing subcutaneous SKOV-3 ovarian carcinoma xenografts. The mice were sacrificed at 4 h after injection. Data are presented in Table 4. The data of normal organs uptake is in excellent agreement with those obtained in normal NMRI mice (Tables 3 and 4). Moreover, efficient tumor targeting has been demonstrated. The tumor uptake of 188Re-ZHER2:V2 (18.7 ± 2.8 %ID/g) exceeded by far the uptake in any other organ or tissue. Tumor uptake exceeded kidney uptake by 5-fold at this time point. As expected, the radioactivity concentration of 188Re-ZHER2:2395 exceeded that of 188Re-ZHER2:V2 in many organs, especially the kidneys (196 ± 38 vs. 3.4 ± 0.3 %ID/g, respectively) and liver (2.5 ± 0.54 vs. 0.40 ± 0.01 %ID/g, respectively). The overall tumor-to-organ ratios are presented in Table 4 for comparison. To evaluate in vivo specificity of HER2 targeting using 188Re-ZHER2:V2, HER2 receptors in tumors were saturated in a group of mice by pre-injection of large excess of non-labeled parental ZHER2:342 affibody molecule. Tumor uptake in this group was 2.2 ± 1.8 %ID/g, which is significantly (p < 0.05) lower than uptake in the non-blocked group (18.7 ± 2.8 %ID/g). The blood concentration (0.18 ± 0.04 %ID/g) did not differ significantly from the concentration in the non-blocked group. The results of this experiment demonstrate saturability of HER2 targeting in vivo and suggest its specificity.

4. Discussion

High renal reabsorption of affibody molecules is the major issue in development of conjugates for radionuclide therapy. The use of residualizing labels results in that the a renal uptake is exceeding the tumor uptake by 15—20 fold, which makes radionuclide therapy non-feasible [16,34,48]. One approach for the development of

Fig. 2. Sensorgram of 188Re-ZHER2:V2 dissociation constants at equilibrium (KD) for binding of radiolabeled affibody molecules to HER2 expressing SKOV-3 cells using LigandTracer Yellow with two different concentrations at 4 °C.

ä ^ O

Re-Z HER2:2395 (-KVDC)


Time, h

Re-ZH 8Re-ZH

in o to as 7 .2

Time, h


Fig. 3. Influence of the amino acid composition and their position in the cysteine-containing chelators on the cellular retention of 188Re-labeled affibody molecules by SKOV-3 cells. A. retention of 188Re-Zher2:2395 (-KVDC), 188Re-Zher2:v2 (-GGGC), 188Re-Zher2:v3 (-GGSC), 188Re-Zher2:v3 (-GSGC); B. retention of 188Re-Zher2:v4 (-GGEC), 188Re-Zher2:v7 (-GEGC), 188Re-ZhER2:V5 (-GGKC), 188Re-Zher2:v8 (-GKGC).

therapeutic conjugates is based on different cellular processing of affibody molecules by tumor cells (slow internalization) and by cell in proximal tubuli in kidneys (rapid internalization). The use of non-residualizing labels provides a rapid clearance of radioactivity from kidneys but slow release from tumors due to a high affinity to the target. This permits obtaining higher radioactivity concentration in tumors than in kidneys and should thus make radionuclide therapy using affibody molecules possible [26,45]. Previous studies have demonstrated that the composition of cysteine-containing peptide-based chelators might be used for modulation of renal uptake of 99mTc and provided higher uptake in tumors than in kidneys [35,36]. The chemical similarity between Re and Tc creates a potential for translation of this approach to the development of therapeutic targeting agents. However, the properties of rhenium and technetium are not identical, and substituting 99mTc with rhenium radioisotopes might appreciably influence the blood clearance rate, as well as the uptake and retention in excretory organs and other tissues [41,45]. This necessitates a re-evaluation of the influence of peptide-based chelators on the uptake and retention of 188Re-labeled affibody molecules in liver and kidneys.

Peptide-based cysteine-containing chelators are directly engineered into the affibody molecules by genetic insertion of peptide-

encoding gene fragments, and the constructs can be produced in high yield in prokaryotic hosts, i.e. E. coli. No further conjugation step is thus required, which simplifies the process and reduces costs. The labeling is site-specific, which is important to provide a uniform radiolabeled product with reproducible physicochemical and biological properties. This is essential, as our previous studies have demonstrated that the position of the chelators noticeably influences the biodistribution and targeting properties of 99mTc-labeled affibody molecules [32].

Generally, labeling with rhenium requires much higher concentration of reducing agents than labeling with technetium, and thus harsher labeling conditions [41]. These conditions are generally non-compatible with proteins that are dependent on disulfide bonds, such as antibodies and antibody fragments. To overcome this, an indirect two-step process was developed for labeling of antibodies where rhenium is first chelated by N3S chelator in harsh conditions, and then the chelator is conjugated to an antibody [49—52]. Multiple steps increase probability of labeling failure and reduces yield. The use of peptide-based chelators permits high-yield labeling (Table 1) of affibody molecules with 188Re in the presence of gluconate as an exchange ligand. Currently, the labeling is performed using a two-vial kit, but it is amenable to development

Table 3

Biodistribution of188Re —labeled affibody molecules in female NMRI mice, 1 and 4 h after intravenous injection. The measured radioactivity of different organs is expressed as % ID/g, and presented as an average value from 4 animals ± SD.

















Blood 1.0 ± 0.1

Lung 1.5 ± 0.1

Liver 2.57 ± 0.25

Spleen 0.7 ± 0.2

Stomach 0.9 ± 0.1

Kidney 148 ± 13

Salivary 0.6 ± 0.1

Muscle 0.30 ± 0.03

Bone 1.1 ± 0.1

Carcassa 9.7 ± 1.3

GI tracta 3.4 ± 0.3

Blood 0.14 ± 0.04

Lung 0.5 ± 0.1

Liver 2.23 ± 0.46

Spleen 0.5 ± 0.1

Stomach 0.55 ± 0.15

Kidney 172 ± 32

Salivary 0.4 ± 0.1

Muscle 0.11 ± 0.01

Bone 0.8 ± 0.3

Carcassa 3.4 ± 0.5

GI tracta 3.1 ± 0.2

1.18 ± 0.08 1.31 ± 0.06 1.6 ± 0.1 1.1 ± 0.3 1.5 ± 0.1 1.2 ± 0.1 1.15 ± 0.12

1.9 ± 0.2 2.1 ± 0.2 2.5 ± 0.2 3.2 ± 0.8 2.0 ± 0.2 2.47 ± 0.45 2.0 ± 0.2

1.3 ± 0.1 2.0 ± 0.2 2.0 ± 0.1 1.8 ± 0.3 1.9 ± 0.2 3.2 ± 0.8 2.6 ± 0.2

0.53 ± 0.04 1.0 ± 0.2 1.1 ± 0.2 1.0 ± 0.3 0.8 ± 0.1 1.25 ± 0.18 1.07 ± 0.07

1.3 ± 0.3 1.38 ± 0.15 2.0 ± 0.2 1.3 ± 0.4 1.5 ± 0.1 2.2 ± 0.6 1.42 ± 0.15

26 ± 6 94 ± 8 68 ± 7 129 ± 21 121 ± 18 150 ± 19 146 ± 8

0.7 ± 0.05 0.8 ± 0.2 1.0 ± 0.1 0.9 ± 0.3 0.9 ± 0.1 1.3 ± 0.3 0.85 ± 0.1

0.33 ± 0.02 0.40 ± 0.02 0.38 ± 0.02 0.35 ± 0.05 0.4 ± 0.1 0.4 ± 0.05 0.4 ± 0.1

0.9 ± 0.3 1.0 ± 0.1 1.1 ± 0.1 1.0 ± 0.2 1.1 ± 0.2 1.16 ± 0.25 0.9 ± 0.2

10 ± 0.3 11 ± 1.0 14 ± 1.3 10.6 ± 1.6 12 ± 1.0 11.3 ± 0.8 10.2 ± 0.5

1.5 ± 0.2 1.9 ± 0.3 2.18 ± 0.35 1.6 ± 0.2 1.8 ± 0.1 2.1 ± 0.2 1.9 ± 0.2

0.15 ± 0.03 0.28 ± 0.05 0.20 ± 0.02 0.2 ± 0.1 0.18 ± 0.05 0.19 ± 0.05 0.20 ± 0.06

0.24 ± 0.09 0.66 ± 0.07 0.40 ± 0.03 1.0 ± 0.3 0.4 ± 0.1 1.0 ± 0.2 0.8 ± 0.1

0.33 ± 0.05 1.44 ± 0.06 0.7 ± 0.1 1.3 ± 0.3 0.8 ± 0.1 3.3 ± 0.5 2.45 ± 0.12

0.16 ± 0.03 0.5 ± 0.1 0.33 ± 0.08 0.47 ± 0.15 0.3 ± 0.1 0.8 ± 0.4 0.8 ± 0.1

0.5 ± 0.3 1.0 ± 0.4 0.7 ± 0.2 0.6 ± 0.4 0.33 ± 0.03 1.5 ± 0.5 0.75 ± 0.20

3.1 ± 0.5 20 ± 4.0 10 ± 1.0 31 ± 6 23 ± 2 152 ± 24 136 ± 23

0.29 ± 0.16 0.56 ± 0.05 0.55 ± 0.06 0.42 ± 0.20 0.25 ± 0.02 0.80 ± 0.35 0.65 ± 0.12

0.05 ± 0.01 0.13 ± 0.04 0.07 ± 0.02 0.09 ± 0.03 0.06 ± 0.01 0.15 ± 0.03 0.12 ± 0.01

0.22 ± 0.05 0.3 ± 0.1 0.21 ± 0.05 0.34 ± 0.07 0.26 ± 0.03 0.6 ± 0.2 0.47 ± 0.04

1.8 ± 0.4 4.1 ± 0.5 2.7 ± 1.2 2.82 ± 0.55 2.3 ± 0.2 5.3 ± 0.9 4.2 ± 0.8

1.89 ± 1.45 1.7 ± 0.6 2.6 ± 0.8 2.5 ± 1.5 1.6 ± 0.5 1.8 ± 0.3 1.4 ± 0.8

Data for gastro-intestinal tract with content and carcass are presented as % of injected radioactivity per whole sample.

of single-vial kit, which would further increase robustness of the procedure [53]. In addition to the small size and rapid kinetics of affibody molecules, their tolerance to harsh labeling conditions (robustness) is a major advantage over immunoglobulin-based targeting agents. All the eight 188Re-labeled affibody molecules bound to HER2-expressing cells with high (low picomolar) affinity (Table 2).

The amino acids adjacent to cysteine had a clear effect on the cellular processing of 188Re-labeled affibody molecules. The cellular retention was the highest for lysine-containing chelators, followed by glutamate- and serine-containing ones (Fig. 3). For each pair of homologous chelators, the GGXC variants (where X = K, E or S) provided better retention of radioactivity than the GXGC counterparts (Fig. 3). This pattern was in excellent agreement with our earlier data concerning cellular processing of 99mTc-labeled variants [36]. As affinities of binding to HER2-expressing cells were very similar for all variants (Table 2), the difference in cellular retention was, most likely, due to difference in cellular processing of radiocatabolites after internalization and lysosomal degradation. This is supported by close resemblance of retention profiles of 188Re-ZHER2:V2 and 188Re-ZHER2:V3 with the profile of the parental affibody labeled with non-residualizing radioiodine labels [54], while retention pattern of 188Re-ZHER2:23g5, 188Re-ZHER2:V5 and 188Re-ZHER2:V8 was similar to the profile of affibody molecules labeled with residualizing 111 In [48]. Despite the lower retention by SKOV-3 cells, the biological half-life of 188Re-ZHER2:V2 and 188Re-ZHER2:V3 was about 15 h, which corresponds well with retention of radioactivity delivered by affibody molecules with non-residualizing radiohalogen labels to HER2-expressing tumors in vivo [26,54].

In vivo data suggest rapid clearance of all conjugates from blood (Table 3). The radioactivity uptake in the gastrointestinal tract (with content) was low, typically less than 2% of injected radioactivity. This suggests that the hepatobiliary pathway played a minor role, if any, in the excretion of the conjugates or their radiometabolites.

The radioactivity uptake in Na/I symporter expressing organs, stomach and salivary gland, was also low. Since these organs accumulate radioperrhenate [55], the low uptake indicates that there was no loss of the radionuclide in vivo due to re-oxidation of 188Re. Uptake in muscle and bone was also low, and variation between conjugates was not substantial. However, the retention of constructs with lysine-containing chelators (i.e. 188Re-ZHER2:23g5, 188Re-ZHER2:V5 and 188Re-ZHER2:V8) was significantly higher than others. The most apparent was the difference in uptakes in lungs, liver, and kidneys (Fig. 4). Uptake of radioactivity at 4 h p.i. was significantly higher for constructs with GGXC variants (where X = E or S) than for GXGC in lung and kidneys. For liver, this was true also for lysine-containing chelators.

The uptake of 188Re-ZHER2:V2 (with GGGC chelator) was lower in all these organs than the uptake of constructs with chelators containing polar or charged side chains. The most striking was the difference in the renal uptake. The renal uptake of 188Re-ZHER2:V2 was 2.5—6 fold lower than uptake of other conjugates already at 1 h p.i. At 4 h p.i., the renal uptake of 188Re-ZHER2:V2 was 40—50 fold lower than the uptake of constructs with lysine-containing chela-tors (188Re-ZHER2:2395, 188Re-ZHER2:V5 and 188Re-ZHER2:V8). Overall, the pattern of the radioactivity retention in kidneys resembled the pattern of cellular retention of the same constructs by SKOV-3 cells, i.e. the ranking of conjugates according to their retention was the same, but the magnitude of the variance was different. Most likely, this difference can be explained by that the internalization rate of affibody molecules in kidneys is much higher than in tumor cells. After re-absorption from primary urine in proximal tubuli, affibody molecules should be degraded in lysosomes. The further fate of radiorhenium depends of physiochemical properties of the radio-catabolites. Most likely, radiocatabolites of 188Re-ZHER2:V2 leak directly into urine, as biodistribution of this agent does not show any site of their possible re-uptake. This creates preconditions for successful targeted radionuclide therapy of HER2-expressing tumors using 188Re-ZHER2:V2.

Liver 1h

Liver 4h

2 2 Q.

Ä2 <o a = 1

Lung 1h

® 2 a

r=n JL

Lung 4h

Hrnflm i

i? 200 Q

Si5 150

ra 100 £1

Kidney 1 h

3? 200 a

sS 150

| 100 tt

3 50 0

Kidney 4h

□ Z2395 (KVDC) □ ZV3 (GGSC) □ ZV4 (GGEC) □ ZV5 (GGKC) ■ ZV2 (GGGC) ZV6 (GSGC) ZV7 (GEGC) ■ ZV8 (GKGC)

Fig. 4. Influence of composition of the —GXYC peptide-based chelator on the uptake of 188Re -labeled affibody molecules in liver, lung, and kidneys 1 and 4 h after injection. The radioactivity of different organs is expressed as % ID/g and presented as an average value from four animals ± standard deviation.

Table 4

Comparative biodistribution oflssRe-ZHER2:V2 andlssRe-Z HER2:2395 in female NMRI nu/nu mice bearing subcutaneous SKOV-3 xenografts at 4 h after intravenous injection. The uptake values are presented as an average value from 4 animals ± SD.

1SSRe-ZHER2:V2 188Re-ZHER2: 2395

Uptake, % Tumor-to-organ Uptake, % Tumor-to-organ

ID/g ratio ID/g ratio

Tumor 18.7 - t2.8b 11.4 t 4.5

Blood 0.20 - t 0.15 92 0.22 0.1 53

Lung 0.39 - 0.01 48 0.8 - t 0.38 14

Liver 0.40 - t 0.01b 46 2.5 0.54 5

Spleen 0.22 - 0.05b 84 0.97 t 0.18 12

Stomach 0.37 0.1 51 0.7 0.25 16

Kidney 3.4 0.30b 5 196 - t 38 0.06

Salivary 0.26 - 0.03b 70 0.61 0.15 19

Muscle 0.09 0.03 210 0.12 0.01 92

Bone 0.21 0.04b 87 0.58 - 0.10 20

Carcass3 2.9 0.62 4.1 0.9

GI tracta b 0.8 - 0.12 2.2 0.3

a Data for gastro-intestinal tract with content and carcass are presented as % of injected radioactivity per whole sample. b Significant difference (p < 0.05) between lssRe-ZHER2:V2 and lssRe-ZHER2:2395.

The biodistribution of 188Re-labeled affibody molecules was very similar to the biodistribution of their 99mTc-labeled counterparts studied earlier [35,36]. However, there were some noticeable differences. For example, the uptake of 188Re-ZHER2:v2 was lower in liver and spleen at 4 h p.i. than the uptake of 99mTc-ZHER2:V2. On the other hand, the uptake of 188Re-ZHER2:V3 was higher in lung, liver, spleen, stomach and salivary gland than the uptake of 99mTc-ZHER2:V3. Despite their chemical similarity, rhenium and technetium labels have different influence on biodistribution of labeled affi-body molecules, necessitating careful structure-properties relationship studies for each label.

The biodistribution of 188Re-ZHER2:V2 in mice bearing HER2-expressing tumor xenografts (Table 4) demonstrated that tumor uptake at 4 h after injection appreciably exceeded uptake in any other organs. In vivo blocking experiment demonstrated HER2 specificity of targeting SKOV-3 xenografts using 188Re-ZHER2:V2. Uptake in blood and bone, which are associated with bone marrow suppression, were 90-fold lower than the tumor uptake. Renal uptake was 5-fold lower that the uptake in tumors. This suggests that 188Re-ZHER2:V2 can efficiently target HER2-expressing tumors and justifies its further development as a targeting agent for radionuclide therapy.

Earlier, we have studied synthetically produced anti-HER2 affibody molecules, maGGG-ZHER2:342 and maGSG-ZHER2:342 labeled with 186Re on the N-terminus [45]. Both 186Re- maGGG and 186Re-maGSG with non-residualizing labels provided low retention of radioactivity in kidneys, making radionuclide therapy possible. However, the placement of the label at the C-terminus in the current study resulted in a 10-fold reduction of hepatobiliary excretion of radioactivity, which reduces the dose to the radiosensitive intestines. Thus, 188Re-ZHER2:V2 is a better format for affibody molecules aimed for therapy.

In conclusion, 188Re-ZHER2:V2 provides lower retention of radioactivity in kidneys, and is a promising candidate for radionuclide therapy of HER2-expressing tumors. This study, along with other studies on affibody molecules, suggests that an optimal labeling strategy permits not only a stable attachment of a radionuclide to a targeting scaffold protein, but also allows for optimization of biodistribution and targeting properties of the radioconjugate.


This research was financially supported by grants from Swedish Cancer Society (Cancerfonden) 2011/483 and 2012/352 and Swedish Research Council (Vetenskapsradet) 2012-163 and 2012-2228.


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