EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to 99mTc-HYNIC labeled avidin and streptavidin

EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to 99mTc-HYNIC labeled avidin and streptavidin

Nuclear Medicine and Biology 39 (2012) 1122–1127 Contents lists available at SciVerse ScienceDirect Nuclear Medicine and Biology j o u r n a l h o m...

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Nuclear Medicine and Biology 39 (2012) 1122–1127

Contents lists available at SciVerse ScienceDirect

Nuclear Medicine and Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n u c m e d b i o

EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to labeled avidin and streptavidin

99m

Tc-HYNIC

Kyung-Ho Jung a, b, Jin Won Park a, Jin-Young Paik a, Cung Hoa Thien Quach a, Yearn Seong Choe a, b, Kyung-Han Lee a, b,⁎ a b

Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Samsung Biomedical Research Institute, Seoul, Korea

a r t i c l e

i n f o

Article history: Received 20 March 2012 Received in revised form 5 June 2012 Accepted 7 June 2012 Keywords: Epidermal growth factor Receptor imaging Streptavidin Avidin 99m Tc

a b s t r a c t Introduction: As direct radiolabeled peptides suffer limitations for in vivo imaging, we investigated the usefulness of radioloabeled avidin and streptavidin as cores to link peptide ligands for targeted tumor imaging. Methods: Human epidermal growth factor (EGF) was site specifically conjugated with a single PEG-biotin molecule and linked to 99mTc-HYNIC labeled avidin-FITC (Av) or streptavidin-Cy5.5 (Sav). Receptor targeting was verified in vitro, and in vivo pharmacokinetic and biodistribution profiles were studied in normal mice. Scintigraphic imaging was performed in MDA-MB-468 breast tumor xenografted nude mice. Results: Whereas both 99mTc-Av-EGF and 99mTc-Sav-EGF retained receptor-specific binding in vitro, the two probes substantially diverged in pharmacokinetic and biodistribution behavior in vivo. 99mTc-Av-EGF was rapidly eliminated from the circulation with a T1/2 of 4.3 min, and showed intense hepatic accumulation but poor tumor uptake (0.6%ID/gm at 4 h). 99mTc-Sav-EGF displayed favorable in vivo profiles of longer circulation (T1/2β, 51.5 min) and lower nonspecific uptake that resulted in higher tumor uptake (3.8 %ID/gm) and clear tumor visualization at 15 h. Conclusion: 99mTc-HYNIC labeled streptavidin linked with growth factor peptides may be useful as a proteinligand complex for targeted imaging of tumor receptors. © 2012 Elsevier Inc. All rights reserved.

1. Introduction The epidermal growth factor (EGF) receptor plays a key role in tumor growth, invasion and metastasis, and has thus become an important target for cancer therapy [1,2]. Accordingly, there is much recent interest in molecular imaging techniques that can monitor tumor EGF receptors in living subjects [3–5]. Although growth factor peptides are the ideal ligand for receptor targeting, the use of directly radiolabeled growth factors for imaging is seriously limited by reduced binding affinity, high in vivo degradation, and rapid blood clearance, which together lead to poor tumor accumulation [6,7]. Our group recently demonstrated that human EGF can be conjugated with a single polyethylene glycol (PEG)-biotin molecule on the N-terminal then linked to streptavidin coated quantum dots, and that this probe displays favorable binding and pharmacokinetic characteristics that lead to high-contrast tumor imaging [5]. A major drawback of using quantum dots as a platform for in vivo use, however, relates to concern over potential toxic effects from the nanoparticle. An obvious way to elude this problem is to render the probe more biocompatible by attaching PEG-conjugated peptides and radiolabel ⁎ Corresponding author. Department of Nuclear Medicine, Samsung Medical Center, Seoul, Korea. Tel.: +82 2 3410 2630; fax: +82 2 3410 2639. E-mail address: [email protected] (K.-H. Lee). 0969-8051/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nucmedbio.2012.06.007

to avidin or streptavidin removed of quantum dots. Avidin, a natural 66–69 kDa protein found in egg whites, and streptavidin, a 60 kDa protein purified from the bacterium Streptomyces avidinii, have remarkably high affinity for biotin (Kd, 10 -13 to 10 -15 M). This has led to their extensive use in a wide-range of biotechnological applications [8–13], including linking of peptide ligands for receptor targeting [11–13]. As an example, a fluorescence-labeled peptide that targets apoptotic cells was attached to streptavidin and successfully used to optically image breast and lung tumors responding to therapy [13]. The choice between streptavidin and avidin, however, is expected to significantly affect the in vivo imaging properties of peptide-based probes. Despite many similarities that are shared, these two proteins significantly differ in primary amino acid sequence, glycosylation state, and electrical charge (Fig. 1) [14–16]. As a consequence, they have been observed to behave quite divergently when administered into living bodies [17–21]. Typically, owing to its high glycosylation and positive charge, avidin is known to clear more rapidly from the circulation [17,18,20] and have greater liver accumulation compared to streptavidin [18–20]. While such behavior can be disadvantageous for imaging, the same physicochemical properties have also been shown to enhance tumor avidin accumulation [22–24]. Furthermore, attachment of PEG and peptide ligands to carrier proteins could affect their in vivo behavior. A better understanding of these issues may support the application of avidin

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to as Av-EGF and Sav-EGF, respectively. 99mTc-Av-EGF and 99mTc-SavEGF was finally purified on a PD-10 column as the first peak fraction.

2.2. Analysis of probe binding to EGF receptor expressing cancer cells MDA-MB-468 human breast cancer cells that overexpress EGFR (kindly provided by Dr. Sang-Min Kim, Samsung Medical Center, Korea) were maintained in 5% CO2 at 37 °C in RPMI 1640 (Lonza; Allendale, NJ) supplemented with 10% fetal bovine serum, 2 mM Lglutamine, 100 U/ml penicillin and 100 mg/L streptomycin. For binding analysis, cells of 80–90% confluence on 100 mm plates were harvested by trypsination, washed with phosphate buffered saline (PBS), and 1 × 10 6 cells were transferred to Eppendorf tubes. Cells were incubated with 54 KBq of 99mTc-Av-EGF or 99mTc-Sav-EGF for 1 h at 37°C in Dulbecco's-PBS and 1% bovine serum albumin. The EGF concentration in each tube for this condition is approximately 127 nM. After incubation, cells were washed twice with 1 ml of cold PBS followed by centrifugation at 1,500 rpm for 1 min, and finally measured for bound radioactivity on a high-energy gamma counter. Binding specificity of the probes was assessed by inhibition of cell binding in the presence of excess cold EGF or the EGFR blocking antibody, cetuximab (Merck Korea).

2.3. In vivo pharmacokinetics and biodistribution studies Fig. 1. Three-dimensional structure of avidin (left) and streptavidin (right) molecules binding biotin (shown as black spheres in the interior).

and streptavidin systems as a universal core to which growth factors can be complexed for receptor-targeted imaging. In this study, we thus investigated the potential utility of 99mTcHYNIC avidin and streptavidin linked with biotin-PEG-EGF for EGF receptor imaging by comparing their pharmacokinetic profile, biodistribution, and tumor imaging characteristics.

2. Materials and methods 2.1. Synthesis and biotin-PEG-EGF

99m

Tc labeling of avidin- and streptavidin-

Human EGF of 6.2 kDa (53 amino acids; Cell sciences, Canton, MA) was site-specifically conjugated to a single biotin-PEG molecule on the N-terminal amine by using slightly acidic conditions as previously described [5]. Briefly, 50 μg of EGF was mixed with biotin-PEG succinimidyl ester (MW 5000; NANOCS; New York) at 1:1 molar ratio in 100 mM sodium acetate (pH 5.5), and incubated overnight at room temperature (RT). Unbound reagents were removed by a protein desalting spin column (mw cutoff, 7000), and the PEGylated product was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For avidin and streptavidin probes, we used commercially available avidin-FITC and streptavidin-Cy5.5 (Invitrogen, Carlsbad, CA), respectively, since these forms can also allow in vivo optical imaging if required. Biotin-PEG-EGF was attached to avidin-FITC or streptavidin-Cy5.5 by 30 min incubation at 1:1 molar ratio in 50 mM sodium-phosphate buffer (pH 8.0) at RT with shaking. Again, unbound reagents were removed by a spin column. For radiolabeling, hydrazinonicotinamide (HYNIC; 1 mg/ml in 20 μl) was first conjugated to biotin-PEG-EGF bound avidin-FITC or strepavidin-Cy5.5 at a ratio of 7.7: 1 by overnight shaking at RT. This was followed by incubation with 74 MBq of 99mTc in 50 μl saline and 12 μl tin-tricine solution at 25 °C for 1 h. For convenience, PEG-EGFlinked avidin-FITC and streptavidin-Cy5.5 will henceforth be referred

All animal experiments were in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC). Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 1.4 to 3.3 MBq of 99mTc-Av-EGF or 99mTcSav-EGF. Blood samples of 5 μl volume were collected from the tail vein at predetermined intervals and measured for radioactivity on a high energy gamma counter and expressed in % injected-dose (%ID) per ml. Time activity curves of blood activity were fitted by nonlinear regression with Graphpad Prism V3.02 software (GraphPad Software Inc., San Diego, CA) using either one- or two-phase exponential decay equations. In the former case, the clearance rate constant (K) and half-life (T1/2) was derived; in the latter, early and late clearance rate constants (K1 and K2) and half-lives (T1/2α and T1/2β) were derived. The area-under-curve (AUC) from zero time to time of last sample was measured with the trapezoid rule, and the AUC from the last data point to infinite time was calculated as the last measured concentration divided by k. Of critical pharmacokinetic parameters, clearance (CL) was calculated as the administered dose divided by the total AUC for the time-concentration relation, while the volume of distribution (Vd) was calculated as the product of T1/2 and CL divided by 0.693. Biodistribution studies were performed in normal ICR mice and MDA-MB-468 tumor bearing Balb/c nude mice following tail vein injection of 18.5 MBq of 99mTc-Av-EGF or 99mTc-Sav-EGF. The amount of EGF included in the administration was approximately 5 μg per mouse (200 μg/kg). Assuming a blood volume of 2 ml, this translates into an immediate blood concentration of 417 nM, which obviously implies a substantially lower concentration in tumor tissue. Tumor models were prepared by subcutaneous injection of 1 × 10 8 MDA-MB468 cells into the right shoulder, and experiments were performed when tumor diameter reached 0.5–1.0 cm. Major organs and tumor were by extracted at 4 h, washed in PBS, weighed, and measured for radioactivity expressed in %ID/gm-tissue. A separate group of tumor bearing mice was evaluated for biodistribution of 37 MBq of 99mTcSav-EGF at 15 h post-injection. The amount of EGF included for administration in these animals was approximately 10 μg per mouse (400 μg/kg).

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2.4. In vivo scintigraphic imaging MDA-MB-468 tumor bearing animals were tail-vein injected with 18.5 MBq of 99mTc-Av-EGF or 99mTc-Sav-EGF and imaged 4 h later, or injected with 37 MBq of 99mTc-Sav-EGF and imaged 15 h later. Animals were anesthetized by intraperitoneal injection of 110 mg/kg ketamine and 9 mg/kg xylazine. Image acquisition was performed on a gamma camera (Trionix Research Laboratory, Ohio) with an intrinsic spatial resolution of less than 3.8 mm, equipped with a 2 mm aperture pinhole collimator. A 15% energy window centered around 140 keV was used, and 15 min (4 h image) or 60 min (15 h image) acquisition data were stored on a 256 × 256 pixel sized matrix. 2.5. Statistical analysis Data are presented as mean±S.D. unless otherwise specified. Student's t-tests were performed to compare cell binding and tissue uptake levels between groups, and p-values of under 0.05 were considered significant. 3. Results Preparation of 99mTc-Av-EGF and 99mTc-Sav-EGF was straightforward and reproducible. SDS-PAGE showed successful monoconjugation of a single biotin-PEG molecule to human EGF. The long biotin-PEG molecule acted to link EGF to avidin or streptavidin, while also serving as a flexible spacer to allow receptor binding without steric-hindrance by the large protein core. Through HYNIC derivatization, biotin-PEG-EGF bound avidin and streptavidin were both efficiently (approximately 30%) radiolabeled with 99mTc, and the final product was readily purified by column chromatography (Fig. 2A & B).

The probes were first evaluated for receptor targeting using high EGF receptor expressing MDA-MB-468 breast cancer cells. The results showed 99mTc-Av-EGF binding to be inhibited 89.5±3.5% by 10 μM cold EGF and 90.1±0.7% by 1.38 μM cetuximab (Fig. 2C). Binding of 99mTc-Sav-EGF was even more severely reduced by 96.6 ±1.0 and 96.2±0.3% with respective treatments (Fig. 2D). The concentration of EGF in the form of radioprobes in these experiments was likely high enough to partly saturate available EGFR binding sites. However, this does not diminish our findings of high specific binding of the probes, since conditions of lower EGF concentration would have increased baseline cell binding, leading to even higher binding specificity. Taken together, these results demonstrate that biotin-PEG mono-conjugated EGF attached to 99mTc-HYNIC labeled avidin and streptavidin retains specific receptor binding properties. The in vivo pharmacokinetic profiles of the probes were then evaluated following intravenous administration into normal mice. 99m Tc-Av-EGF was shown to follow a mono-exponential mode of blood clearance with a rate constant (K) of 0.161 and half-life of 4.3 min (Fig. 3A). The volume of distribution (Vd) was 4.7 ml, and clearance (CL) was 45.5 ml/min. 99m Tc-Sav-EGF displayed a completely divergent pharmacokinetic pattern and was cleared from the circulation in a bi-exponential manner. The early (K1) and late rate constants (K2) of 0.377 and 0.019 led to early distribution (T1/2α) and late clearance half-lives (T1/2β) of 2.1 min and 51.5 min, respectively (Fig. 3B). The Vd for 99mTc-Sav-EGF was 0.79 ml and the CL was 0.64 ml/min. These results reveal a substantially slower rate of elimination with a longer duration of retention in the circulation for 99mTc-Sav-EGF compared to 99mTc-Av-EGF. The 4 h biodistribution of intravenously injected 99mTc-Av-EGF and 99mTc-Sav-EGF in normal mice is summarized on Table 1. Consistent with the pharmacokinetic findings, blood activity

Fig. 2. Column chromatography profiles and cell binding characteristics. (A & B) Radioactivity of fractions collected following elution of 99mTc-Av-EGF (A) and 99mTc-Sav-EGF (B) through a PD-10 column. Radiolabeled probes are included in the 1st peak. (C & D) Binding of 99mTc-Av-EGF (C) and 99mTc-Sav-EGF (D) to MDA-MB-468 breast cancer cells. Receptor specificity of binding was assessed by competition with excess cold EGF or blocking antibody (Cetuximab). Bars are mean±S.D. expressed as % relative bound activity obtained from a single representative experiment of two separate experiments performed in triplicate. kcpm, kilo-counts per min; †, Pb.005; ‡, Pb.001, compared to control group.

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In vivo scintigraphy acquired 4 h after 99mTc-Av-EGF injection displayed intense liver uptake with poor tumor visualization (Fig. 5A). In comparison, 99mTc-Sav-EGF imaging at 4 h showed better tumor visualization, although with only modest contrast (Fig. 5A). When imaging time was delayed to 15 h (with an increased dose), highcontrast tumor imaging was achieved for 99mTc-Sav-EGF (Fig. 5B). 4. Discussion

Fig. 3. In vivo pharmacokinetic profiles of EGF probes. Nonlinearly fitted time-activity curves of radioactivity measured from blood samples following intravenous-injection of 99mTc-Av-EGF (A) or 99mTc-Sav-EGF (B). Curves were analyzed for pharmacokinetic parameters of rate constant (K), half-life (T1/2), volume of distribution (Vd) and clearance (CL). Data are mean±S.D. of % initial activity obtained from 5 ICR mice per group.

remaining at 4 h was 5-fold higher for 99mTc-Sav-EGF compared to 99m Tc-Av-EGF. In contrast, 99mTc-Av-EGF had greater accumulation in the liver, which likely reflects nonspecific binding from its basic charge and high glycosylation. 99mTc-Av-EGF also had slightly higher uptake in the kidneys. Activity in other tissues including skeletal muscle, heart, and lungs were similarly low for both probes. Stomach activity of both probes was also low, indicating absence of significant free 99mTc release. The probes were then intravenously injected into MDA-MB-468 tumor bearing nude mice. Biodistribution 4 h following administration displayed significantly higher tumor uptake of 99mTc-Sav-EGF (1.62±0.35 %ID/gm; Fig. 4A) compared to 99mTc-Av-EGF (0.62±0.11 %ID/gm; P=.0016; Fig. 4B). Tumor uptake of 99mTc-Sav-EGF further significantly increased by 15 h to reach 3.78±0.17%ID/gm, which was 5.1-fold greater activity than that of skeletal muscle (Fig. 4B).

Table 1 Biodistribution of 99mTc-Av-EGF and intravenous injection. 99m

Blood Heart Lung Liver Spleen Stomach Kidney Muscle

99m

Tc-Av-EGF (n = 4)

0.78±0.32 0.54±0.10 0.58±0.17 24.96±3.57 6.67±1.46 0.69±0.02 12.79±1.19 0.16±0.01

Tc-SAv-EGF in normal mice at 4 h after 99m

Tc-SAv-EGF (n = 4)

4.08±0.48 0.77±0.07 1.05±0.11 9.21±0.55 5.54±0.46 0.78±0.12 7.12±0.83 0.21±0.02

Data are mean±S.D. of % injected-dose per gram-tissue.

P .000 .003 .001 .000 .113 .207 .000 .001

The use of directly radiolabeled peptides for in vivo imaging suffers major drawbacks including diminished target affinity, susceptibility to proteolytic degradation, and excessively rapid blood clearance. These problems can potentially be alleviated and imaging properties made more favorable by attaching peptide ligands to a larger protein core. In this study, we exploited the strong bond between avidin/ streptavidin and biotin to attach EGF into a ligand-protein complex for targeted imaging. As a result, both avidin and streptavidin readily attached mono-conjugated biotin-PEG-EGF in a manner that retained receptor-specific binding, and were efficiently labeled with 99mTcHYNIC. However, in vivo studies revealed a substantial divergence in pharmacokinetic profile and biodistribution of the EGF probes depending on the choice between avidin- and streptavidin-core. In our pharmacokinetic studies, intravenously injected 99mTc-AvEGF rapidly cleared from the circulation in a mono-exponential pattern with a half-life of b5 min, which led to less than 10% of administered dose in 1 ml of blood by 10 min. In contrast, 99mTc-SavEGF displayed a substantially slower bi-exponential clearance pattern with more than 25% of the injected dose remaining in 1 ml blood after 4 h. Despite their resemblance in tetravalent high affinity binding to biotin, avidin and streptavidin actually only share 38% homology in amino acid sequence. Avidin has twice the number of lysine and arginine residues that gives it a net positive charge, and also has a high level of glycosylation [14–16]. Together, these physicochemical properties are known to contribute to a rapid rate of elimination of avidin from the circulation [17,18,20]. In contrast, streptavidin has a near-neutral pI and lacks any carbohydrate modification, which help prolong its blood retention in vivo [17,18,20]. Our results demonstrate that these blood clearance profiles are largely preserved following attachment of long PEG molecules, EGF peptides and 99mTc-HYNIC. The biodistribution of the two EGF probes partly recapitulated previous observations for free avidin and streptavidin. Hence, at 4 h in normal mice, 99mTc-Av-EGF demonstrated high hepatic and renal uptakes that exceeded 20 and 10 %ID/gram-tissue, respectively. High liver accumulation can be attributed to carbohydrate moieties of avidin that bind to sugar receptors present on liver Kupffer cells [9,20]. Elevated kidney uptake is likely due to the positive net charge that bind to negatively charged renal glomerular basement membrane [9,20]. In comparison, 99mTc-Sav-EGF showed significantly lower accumulation in the liver (2.7-fold). Accumulation in the kidneys was also lower for 99mTc-Sav-EGF, but to a lesser extent (1.8fold). The reason for the smaller difference in renal uptake is not clear. However, a previous study interestingly reports that truncated forms of streptavidin that can occur through proteolytic degradation have exceptionally high affinity for the kidneys [19]. It is therefore possible that the presence of such forms in some of the 99mTc-Sav-EGF molecules contributed to the higher than expected renal accumulation seen in our study. Low radio-uptakes by the stomach and thyroid indicate low release of free 99mTc for both tracers. In addition, the strong binding of biotin to streptavidin and avidin, which remains highly stable against a wide range of pH, heat, and proteolytic enzymes, favors the stability of the tracers in our experiments. Nonetheless, since we did not directly check for stability in our study, partial dissociation of the bond cannot be completely excluded, and this could potentially have influenced the biodistribution pattern of our tracers. Furthermore, as streptavidin by itself can show nonspecific tumor uptake [25],

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Fig. 4. Biodistribution and tumor uptake of EGF probes in tumor bearing mice. Biodistribution of 99mTc-Av-EGF at 4 h (A) or 99mTc-Sav-EGF at 4 and 15 h (B) following intravenous injection into MDA-MB-468 tumor bearing mice. Data are mean±S.D. of % injected dose per gram-tissue (% ID/gm) obtained from 4 animals per group. *, Pb.05, †, Pb.005; ‡, Pb.0001, when comparing 15 h to 4 h activity.

additional experiments with radiolabeled avidin and streptavdin that do not contain EGF as control would have been useful to support the specificity of tumor uptake of our tracers. These issues should be considered limitations of our study. Owing to the more favorable kinetic and distribution properties, 99m Tc-Sav-EGF showed significantly higher tumor uptake compared to 99mTc-Av-EGF in tumor bearing mice. The advantages of 99mTcSav-EGF were also demonstrated on scintigraphic experiments where high-contrast tumor images were shown, compared to the

prominent liver and kidney uptake with poor tumor visualization displayed for 99mTc-Av-EGF images. The avidin-biotin system offers a versatile tool for in vivo targeting purposes, which has mostly been centered on pretargeting strategies. Our study demonstrates that the system can also be used to increase delivery of peptide probes to target tissue through improved in vivo pharmacokinetics. A major hurdle for the clinical translation of this approach, however, is that it may be difficult to repeatedly administer streptavidin without eliciting an immune response. Therefore, newer techniques such as site-directed mutation [26] may be required before this technique can be applied to human subjects. In conclusion, biotin-PEG mono-conjugated EGF can be conveniently linked to 99mTc-HYNIC labeled avidin- and streptavidin-core as receptor targeting probes. Among the two types of probes, 99mTcSav-EGF displayed more favorable in vivo profiles of longer circulation and lower nonspecific uptake that resulted in clear tumor delineation. Thus, 99mTc-HYNIC labeled streptavidin linked with growth factor peptides may be useful as a protein-ligand complex for targeted imaging of tumor receptors. Acknowledgments This work was supported by the Korean Science and Engineering Foundation (KOSEF) Grant funded by the Korea Government (# 20110002288).

Fig. 5. Scintigraphic imaging in MDA-MB-468 tumor bearing mice. Animals were intravenously injected with 17.9 MBq 99mTc-Av-EGF (A) or 99mTc-Sav-EGF (B, left) and imaged 4 h later for 15 min, or injected with 37 MBq 99mTc-Sav-EGF and imaged 15 h later for 60 min (B, right). Anesthesia for imaging was performed by intraperitoneal injection of 110 mg/kg ketamine and 9 mg/kg xylazine. Arrows indicate subcutaneous tumor.

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