[ 177 Lu]Bz-DTPA-EGF: Preclinical Characterization of a Potential Radionuclide Targeting Agent Against Glioma

CANCER BIOTHERAPY & RADIOPHARMACEUTICALS Volume 19, Number 2, 2004 © Mary Ann Liebert, Inc.

[177Lu]Bz-DTPA-EGF: Preclinical Characterization of a Potential Radionuclide Targeting Agent Against Glioma Åsa Liljegren Sundberg, Lars Gedda, Anna Orlova, Alexander Bruskin, Erik Blomquist, Jörgen Carlsson, and Vladimir Tolmachev Division of Biomedical Radiation Sciences, Uppsala University, Uppsala, Sweden

ABSTRACT Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T1/ 2 5 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177 Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cellbinding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPAEGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cellassociated after 2 days. Biodistribution studies of i.v. injected [177 Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo. Key words: glioma, EGF, Bz-DTPA, Lutetium-177, biodistribution, cellular processing, radionuclide therapy INTRODUCTION Targeted radionuclide therapy is a treatment modality based on the selective delivery of cytotoxic radionuclides to tumor cells. Tumor-associated antigens on the cellular membrane or overexAddress reprint requests to: Åsa Liljegren Sundberg; Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University; S-751 85 Uppsala, Sweden; Tel.: 146-18-471-38-68; Fax: 146-18-471-34-32 E-mail: [email protected]

pressed cell-surface receptors may be used as targets to find the malignant cells.1 Monoclonal antibodies and their fragments, as well as signaling peptides and their analogues, may be utilized for targeting. After many years of trials and errors, targeted radionuclide therapy has now been established as a clinical option for treatment of hematological malignancies, such as non-Hodgkin’s lymphoma.2,3 Moreover, a number of clinical trials have demonstrated encouraging results in the treatment of solid neuroendocrine tumors using radiolabeled somatostatin analogues.4–6 A 195

reason for the successful outcome of these trials may be the use of short peptides as targeting agents. Due to their small size, these molecules can ensure fast tumor penetration and quick blood clearance, thereby providing a low dose burden to healthy organs and tissues. Targeted radiotherapy could be of great benefit for patients with malignant glioma, glioblastoma multiforme (GBM). GBM almost never metastasizes outside the central nervous system, but local recurrences in the brain limit survival to only a few months. Locoregional radioimmunotherapy of gliomas has been shown to prolong survival, but a curative effect has not been achieved, possibly due to the limited diffusion of bulky antibodies in the brain.7,8 The use of radiolabeled somatostatin analogues, however, enabled the targeting of distant foci of low-grade gliomas and gave promising therapeutical results, indicating an advantage of using small, diffusible peptides also for brain tumor treatment.9–11 Unfortunately, the expression of somatostatin receptors is low in high-grade gliomas. The epidermal growth factor receptor (EGFR) is overexpressed in a high percentage of the glioblastomas—especially in primary glioblastomas developed in older patients—and it might, therefore, be used for the targeting of GBM.12–15 The EGFR is a transmembrane tyrosine kinase receptor that is normally regulated by the binding of its ligands (e.g., epidermal growth factor [EGF]), or transforming growth factor-a (TGF-a) to the extracellular domain of the receptor. This triggers receptor dimerization, autophosphorylation of the intracellular tyrosine kinase, and the subsequent recruitment and phosphorylation of other substrates. These substrates stimulate a number of downstream signal transduction pathways that promote cell proliferation and survival.16 Overexpression of EGFR is thought to contribute to the malignant phenotype of human GBMs. The ligand, EGF, is a relatively small, 53-amino-acids-long peptide, which binds to its receptor with subnanomolar affinity.17 The small size of EGF may provide an advantage in tumor targeting in comparison with large antibodies, even though it is not as small as the somatostatin analogues. Further progress in therapeutic radionuclide targeting might be achieved by choosing radionuclides with appropriate nuclear properties. Theoretical calculations have demonstrated that the probability for the cure of a tumor of a given size depends on the path length of the beta-particle, emitted by the therapeutic nuclide.18,19 One problem with radionuclide therapy of small tu196

mors is that the radiation energy emitted is absorbed by surrounding tissue rather than by the tumor cells. With larger tumors, the obvious problem instead is the large number of tumor cells. This implies that, for patients with tumors of various sizes, an improved treatment might be achieved by using a panel of several radionuclides, a “radionuclide cocktail” approach. A recent animal study demonstrated that radionuclide therapy with a combination of 90Y- and 177Lu-labeled DOTATATE (a somatostatin analogue) was superior to radionuclide therapy with 90Y- or 177 Lu-labeled DOTATATE only.20 When selecting a therapeutic label for EGF, we considered that a possible glioma patient might be burdened with a bulky residual tumor, somewhat smaller tumor cell clusters, and spread single cells. Such a variety of targets suggest that the “radionuclide cocktail” approach may be an advantage in the treatment. The Auger and conversion electron emitter 111In (T1/2 5 2.83 days), the lowenergy beta emitter 177Lu (T1/2 5 6.71 days, Ebmax 5 497.1 keV), and the high-energy emitter 90Y (T1/2 5 2.67 days, Ebmax 5 2281.4 keV) may be used. The idea is to use the same targeting agent, a benzyl-DTPA-EGF conjugate, as the carrier for all three radionuclides. [111In]Bz-DTPA-EGF may be a good targeting agent for single tumor cells, since the range of the electrons emitted from 111In is quite short (0.02–10 mm for Auger electrons and 200– 500 mm for conversion electrons). For the therapy of bulky residual tumors, however, it may be preferable to use a radionuclide with a larger particle range, such as 90Y, which is frequently used in the clinic. The low-energy b particle emitter 177Lu should be more suitable than 90Y for the treatment of small tumor cell clusters. 177Lu also emits gamma radiation (208 keV, 11%), with an energy suitable for scintigraphy and dosimetry. Preclinical characterization of [111In] Bz-DTPA-EGF, designed for eradication of spread single cells, has been reported elsewhere.21 The goal of this study was to prepare and preclinically characterize the [177Lu]Bz-DTPA-EGF conjugate both in vitro and in vivo, in order to evaluate its targeting properties.

MATERIALS AND METHODS Materials 177Lu was obtained from IDB Holland (Baarle Nas-

sau, The Netherlands). All commercially available chemicals were of p.a. grade or better. High-qual-

ity (resistance 18 MOhm/cm3) Milli-Q water (Millipore, Billerica, MA) was used for the preparation of all solutions. Disposable NAP-5 size exclusion columns were obtained from Pharmacia (Uppsala, Sweden). Recombinant human epidermal growth factor, hEGF (Chemicon, Temecul, CA), was used in all experiments. Chloramine-T (CAT) was from Sigma Chemical Company (St. Louis, MO). NCSBz-DTPA was synthesized in our laboratory in accordance with a previously reported method.22 During cell experiments, cells were seeded in Petri dishes obtained from Nunc, Roskilde, Denmark. Cells were counted with a Coulter Z2 cell counter (Beckman Coulter, Fullerton, CA). Radiolabeling An EGF solution in 0.1 M borate buffer, with a pH of 9.1 (8 mL, 40 mg), was added to a solution of NCS-benzyl-DTPA in the same buffer (50 mL, 1 mg/mL), and the mixture was shaken overnight. Next morning, 1 mL of borate buffer was added, and the mixture was passed through a SPEC C18 (Ansys Diagnostics, Inc., Lake Radiolabeling, Forest, CA) column. The column was subsequently washed with 0.5 mL of borate buffer, with a pH of 9.3, and 0.5 mL of 5% acetonitrile in water, and the Bz-DTPA-EGF conjugate was eluted with 0.5 mL of 50% acetonitrile in water. For the buffer change, the eluate containing Bz-DTPA-EGF was loaded onto a NAP-5 column preequilibrated with 0.1 M of acetate buffer, with a pH of 6.0. Separation was performed according to the manufacturer’s instructions using acetate buffer, and the high-molecularweight fraction was used for labeling. A stock solution of 177Lu in 0.02 N HCl (5–20 MBq) was added to 6 mg of Bz-DTPA-EGF in 0.1 M of ammonium acetate (pH 6.0). The reaction mixture was incubated for 1 hour at room temperature. The labeled conjugate was purified on a NAP-5 column using PBS. The labeled products were additionally analyzed using SPEC C18 columns.

Chemie, Taufkirchen, Germany), L -glutamine (2 mM), and PEST (penicillin 100 IU/mL and streptomycin 100 mg/mL), both from Biochrom Kg, in Germany. In all cell experiments, cells were grown at 37°C in incubators with humidified air, equilibrated with 5% CO2. During culture and experiments, cells were trypsinized with trypsinEDTA (0.25% trypsin, 0.02% EDTA in PBS without Ca and Mg) from Biochrom Kg, in Germany. Binding of [177Lu]Bz-DTPA-EGF to U343 Cells The binding of [177Lu]Bz-DTPA-EGF to U343 glioma cells, cultured in 3.5-cm Petri dishes, was evaluated, and discrimination was made between membrane-bound and internalized radioactivity. Typically, the cells (about 6 3 105 cells/dish) were washed once with a culture medium, and then incubated with 1 mL [177Lu]Bz-DTPA-EGF solution (about 2.6 ng per dish in culture medium). In order to determine the amount of unspecific binding, an excess of unlabeled EGF (1 mg) was also added to some dishes. After 0.5–24 hours, the dishes were washed six times with a cold, serum-free medium, and then treated with 0.5 mL of ice-cold 0.1 M glycin-HCl buffer, with a pH of 2.5, for 6 minutes at 0°C, in order to remove membrane-bound radioactivity. An additional 0.5 mL of the glycin-HCl buffer was used to wash the cells once. In order to collect the remaining internalized radioactivity, the cells were treated with 0.5 mL of 1 M NaOH solution at 37°C for about 90 minutes. Another 0.5 mL of NaOH solution was used for washing. The collected fractions were measured in an automated gamma counter. Dishes treated with an excess of EGF were not treated with acid and base, but were instead trypsinized with 0.5 mL of trypsin-EDTA. Part of the cell suspension was then used for cell counting (0.5 mL), and the remaining 1 mL was measured in the gamma counter.

Cell Culture

Retention of Radioactivity after Interrupted Incubation with [177Lu]Bz-DTPA-EGF

The human glioma cell line U343MGaC12:6 (from now on denoted U343) that has a high expression of EGFR, approximately 5 3 105 EGFR per cell, was used in all cell experiments. The cell line was established in Uppsala by Westermark et al. in 1982,23 and is regularly screened for mycoplasma. The cells were cultured in Ham’s F10 medium (Biochrom Kg, Berlin, Germany) supplemented with 10% fetal bovine serum (Sigma,

The retention of radioactivity in U343 cells after interrupted incubation with [177Lu]Bz-DTPAEGF was investigated. U343 cells (about 6 3 105 cells/dish) cultured in Petri dishes (3.5-cm diameter) were incubated with 1 mL of [177Lu]BzDTPA-EGF solution (about 2.6 ng per dish in culture medium). After 5 hours, the cells were washed six times with a cold, serum-free medium in order to remove unbound conjugate, and the 197

incubation was then continued in a cell culture medium. At the indicated time points (0–45 hours), the cells were washed six times and treated with solutions of glycin-HCl and NaOH as described above. The collected fractions were measured in an automated gamma counter. Additional dishes were used for cell counting. Saturation Assay, Determination of the Dissociation Constant, Kd U343 cells cultured in 24-well plates (about 1 3 105 cells/well) were placed on ice and washed once with a cold, serum-free medium. A dilution series of [177 Lu]Bz-DTPA-EGF (100 to 0.4 ng/ mL) was added to the wells, and the plates were incubated on ice for 4 hours. For every conjugate concentration, there was also a blocked control containing 1003 molar excess of EGF. After the incubation, the cells were washed six times with a cold, serum-free medium and trypsinized with 0.5 mL of trypsin-EDTA. After mixing the cells with 1 mL of culture medium, a part of the cell suspension (0.5 mL) was used for cell counting, and the remaining 1 mL was measured in an automated gamma counter. Biodistribution of Intravenously Injected [177Lu]Bz-DTPA-EGF in Mice The in vivo studies were carried out in adult female NMRI mice (26–35 g) (Möllegård, Ry, Denmark) in a controlled environment. All animals were handled according to guidelines by the Swedish Animal Welfare Agency, and the experiments were approved by the local Ethics Committee for Animal Research. Groups of 4 mice per timepoint were injected intravenously (i.v.) via the tail vein with 50 mL of [177Lu]BzDTPA-EGF solution (100 kBq, 200 ng in PBS) per animal. The animals were sacrificed at 0.5, 1, 4, and 24 hours post injection. They were anesthetized by an intraperitoneal (i.p.) injection of a mixture of Rompun (1 mg/mL) and Ketalar (10 mg/mL), 0.2 mL per 10 g of animal weight, and killed by heart puncture. Blood, urine, and feces was collected, and the heart, bladder, pancreas, spleen, stomach, liver, kidneys, lungs, small and large intestine, skin, muscle, bone, thyroid, salivary glands, brain, and tail were dissected, weighed, and measured for radioactive content with an automated gamma counter. Organ values were calculated as a percent of injected amount per g of organ (% ID/g).

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Blocking EGFR Specific Binding of [177Lu]Bz-DTPA-EGF in Mice The receptor-specific uptake of [177Lu]Bz-DTPAEGF in vivo was evaluated in 8 normal NMRI mice. Four of the animals were first i.v. injected via the tail vein with an excess of human recombinant EGF (100 mg in 50 ml PBS per animal). All animals were then injected with 50 mL of [177Lu]Bz-DTPA-EGF solution (100 kBq, 200 ng in PBS). The animals were sacrificed 0.5 hours post injection of [177Lu]Bz-DTPA-EGF, as described above. The collected organs were weighed and measured for radioactive content with an automated gamma counter, and organ values were calculated as above.

RESULTS In vitro Experiments The binding of [177Lu]Bz-DTPA-EGF to U343 cells is shown in Figure 1. At all timepoints tested, the vast majority of the cell-associated radioactivity was internalized. Figure 2 shows that the binding of the conjugate to glioma cells was EGFR-specific, since it could be displaced by an excess of unlabeled EGF. The retention of radioactivity after interrupted incubation with [177Lu]Bz-DTPA-EGF is shown in Figure 3. About 65% of the radioactivity was still cell-associated after 45 hours, and a majority of this was internalized. Figure 4 shows the result of the saturation assay with [177 Lu]Bz-DTPA-EGF. The specific binding in pmol/105 cells is plotted against the total molar concentration of added [177Lu]BzDTPA-EGF, and the result was analyzed by nonlinear regression using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). The obtained Kd value was 1.9 nM. Biodistribution and Blocked Binding in vivo The kinetics of 177 Lu radioactivity in blood after an i.v. injection of [177Lu]Bz-DTPA-EGF in mice is shown in Figure 5. The conjugate shows quick blood clearance, with less than 1% ID/g of whole blood 1 hour post injection. The biodistribution of [177Lu]Bz-DTPA-EGF in normal mice is shown in Figure 6. A majority of the radioactivity was, at all timepoints, found in the liver, kidneys, and spleen (up to about 35%,

Figure 1. Binding of [177Lu]Bz-DTPA-EGF to cultured U343 glioma cells. The cell-associated radioactivity as a function of time, discriminated into membrane-bound (filled diamonds), internalized (crosses) and total cell–associated radioactivity (open circles). The presented data are mean values of three measurements, and the error bars represent the standard deviation.

24%, and 9% ID/g, respectively). Accumulation of radioactivity at a lower level was also seen in the pancreas, stomach, lungs, small and large intestine, bone, thyroid, and submaxillary gland. Virtually no uptake was seen in the brain. For all organs except bone, the overall tendency was that the radioactivity accumulation decreased over

Figure 2. Specificity of [177Lu]Bz-DTPA-EGF binding at three time points. In the blocked samples, excess amounts of unlabeled EGF was added in order to block the EGFRs prior to the addition of [177Lu]Bz-DTPA-EGF. The presented data are mean values of three measurements, and the error bars represent the standard deviations.

time. In bone, the radioactivity seemed to increase with time, although at a rather low level (up to about 2.9% ID/g). Radioactivity in urine and feces was measured at each data point, but there was no opportunity to collect whole urine and feces during this study. For this reason, information about the excretion pathways is of a semiquantitative nature. We could, however, observe that a great amount of the 177Lu radioactivity is located in the urine already at earlier timepoints, 0.5–1 hour postinjection. Urinary excretion continues to play an important role in radioactivity elimination throughout the whole observation period. In feces, 177 Lu radioactivity appeared at 1 hour postinjection, and there were appreciable amounts also at later timepoints, 4 and 24 hours (data not shown). In order to elucidate which organs had a receptormediated uptake of labeled conjugate, biodistribution was measured at 30 minutes with and without preinjection of a large amount of nonlabeled EGF. The data are shown in Figure 7. The uptake in the liver and kidneys (Figure 7A) is shown separately from the low-uptake organs (Figure 7B). The figure indicates that radioactivity uptake in the liver could be reduced by EGF. The same decrease in uptake could also be seen in some other organs, such as the pancreas, stomach, small and large intestine, and submaxillary gland. In the pancreas, as much as about 80% of

Figure 3. The cellular retention of radioactivity as a function of time, divided into membrane-bound (filled diamonds), internalized (crosses) and total cell–associated radioactivity (open circles). The cells were incubated with [177Lu]BzDTPA-EGF for 5 hours and were then washed before the retention incubation in fresh cell-culture medium started. The presented data are mean values of three measurements, and the error bars represent the standard deviations.

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Figure 4. Saturation study of [177Lu]Bz-DTPA-EGF. Different amounts of the conjugate were allowed to bind cultured U343 cells during 4 hours incubation on ice. The data were analyzed by GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). All data points are mean values of at least 3 data points, and maximal variations are shown.

the binding could be blocked. In the kidneys, on the other hand, blocking with EGF led to an increased accumulation of radioactivity.

DISCUSSION We have previously evaluated the targeting capacities of an 111In-labeled Bz-DTPA-EGF conjugate in vitro and the results showed a high, specific uptake of [111In]Bz-DTPA-EGF in cultured glioma cells. 21 Because very promising results were obtained in tumor treatment by 177Lu-labeled peptides, both in animal models24,25 and in patients, 26 this nuclide was included into a panel of possible therapeutic labels for EGF-based antiglioma conjugates. An experience with somatostatin analogues demonstrated that the change of nuclide in the same chelator may affect the tumor-targeting properties of a peptidebased conjugate.27,28 This, however, was not the case when substituting 111In for 177Lu in BzDTPA-EGF. The uptake in glioma cells in vitro was high and specific and the obtained dissociation constant, (K d 5 1.9 nM), for the [177Lu]BzDTPA-EGF conjugate, was very similar to that obtained with the 111In-labeled conjugate (2.0 nM). The [177Lu]Bz-DTPA-EGF conjugate was also rapidly internalized into the tumor cells. This is favorable for therapy because the radionuclides are then situated closer to the cell nucleus, and

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the radiation dose to DNA thereby appreciably increases. 29 Another encouraging result was the good retention of radionuclides in tumor cells. About 65% of the radioactivity was still cell-associated after 45 hours, which is close to the value for the corresponding 111In-conjugate (59 6 1.5% after 45 hours). A good cellular retention of radioactivity is vital in targeted therapy, since it increases the dose to tumor tissue and thereby also increases the chance to permanently inactivate the tumor cells. It also reduces the dose to normal tissues that otherwise might accumulate free radionuclides or other degradation products. It is known that normal cells in some tissues (e.g., hepatocytes in the liver) express EGFRs to some extent.30 We, therefore, used these organs as a model to investigate the [177Lu]Bz-DTPA-EGF conjugate’s capability to target EGFR in vivo. To confirm that the organ uptakes were receptor-specific, some mice were injected with excess amounts of unlabeled EGF prior to an injection of [177Lu]Bz-DTPA-EGF to block the receptors. The results showed that the targeting was probably receptor-specific in the liver, pancreas, stomach, small and large intestine, and submaxillary gland. Expression of EGFRs in these organs has been reported earlier.31–33 Thus, it could be concluded that the conjugate was able to target EGFRs in vivo. In the kidneys, however, a significant increase— rather than decrease—in uptake could be seen in the blocked mice. This was probably due to a shift in the excretion pathway towards renal excretion, caused by the blocking of the liver.

Figure 5. Blood kinetics of [177Lu]Bz-DTPA-EGF in mice. The presented data points are mean values from four animals, and the error bars represent the standard deviations.

Figure 6. Biodistribution of [177Lu]Bz-DTPA-EGF in normal mice. The presented data are mean values of measurements from four animals, and the error bars represent the standard deviations.

The expression of EGFRs in normal tissue is obviously a considerable problem with EGFR targeting. Systemic administration of radiolabeled EGF is, therefore, most likely not an option for targeted therapy. When targeting glioblastomas, however, this problem may be circumvented by using a locoregional intracranial administration. A majority of the radiolabeled conjugate will then, hopefully, remain in the brain and not be released in the blood circulation. This way of administrating tumor-targeting agents, most often antibodies, has been used in several studies, with relatively small leakage to the cerebrospinal fluid or the blood.5,8 However, leakage of radiolabeled substance into the blood may occur, especially because glioblastoma patients often have a disrupted blood–brain barrier due to their disease. 34 It is, therefore, important to investigate the biodistribution of systemically injected [177 Lu]Bz-DTPA-EGF conjugate, in order to estimate the risk of radiation exposure to critical organs. The results of our study showed that

two organs—the liver and kidneys—at all timepoints accumulated the majority of the radioactivity. The highest uptake per g of tissue was found in the liver. There might, however, be a positive aspect of the liver accumulation of circulating conjugate; the use of hepatic clearance has earlier been proposed in order to improve tumor-to-nontumor localization ratios of radiolabeled antibodies for radioimmunodetection and radioimmunotherapy, utilizing the ability of hepatocytes to excrete degradation products into the bile.35–38 Thus, removal of radioactivity from blood circulation through the liver might spare more radiosensitive tissues, like bone marrow and the kidneys, from high radiation doses. An encouraging sign was that the uptake in almost all organs decreased with time. An exception was bone, where the radioactivity accumulated with time. Bone marrow is often a dose-limiting organ in radionuclide therapy, and whether this causes problems in our case needs to be analyzed. Earlier studies have indicated that the reason for

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A

60

non-blocked blocked

Uptake (% ID/g)

50 40 30 20 10 0 liver

kidneys

Figure 7. Influence of preinjection of non-labeled EGF on the biokinetics of [177Lu]Bz-DTPA-EGF. Uptake in the liver and kidneys is shown in (A) and the other organs in (B). The presented data are mean values of measurements from four animals, and the error bars represent the standard deviations.

small intestine

Uptake (% ID/g)

B

such an accumulation might be the release of lanthanides from acyclic chelators at acidic lysosomal pH, with subsequent accumulation in the bone matrix.39,40 The accumulation of radionuclides in bone might, therefore, be an indication that the in vivo stability of [177Lu]Bz-DTPA is not optimal. The use of macrocyclic chelators, such as DOTA derivatives, might solve this problem, and we are currently investigating the possibilities to use these chelators for EGF-based conjugates. 202

CONCLUSIONS In conclusion, [177Lu]Bz-DTPA-EGF is a potential antiglioblastoma-targeting conjugate that binds EGFR with good affinity and high specificity, both in vitro and in vivo. It is also rapidly internalized and has excellent cellular retention, which is advantageous for radionuclide-targeting therapy. Results of the biodistribution study showed the ability of the conjugate to target EGFR in vivo.

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