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A novel tumor pretargeting system based on complementary L-configured oligonucleotides Maik Schubert, Ralf Bergmann, Christian Foerster, Wiebke Sihver, Stefan Vonhoff, Sven Klussmann, Lucas Bethge, Martin Walther, Jörn Schlesinger, Jens Pietzsch, Jörg Steinbach, and Hans-Juergen Pietzsch Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.7b00045 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Bioconjugate Chemistry

Cetuximab

linker

5‘CCG

linker

CAC CGC ACA GCC GC3‘

5‘GCG

GCT GTG CGG TGC GG3‘ 10kDa PEG

24 h

ACS Paragon Plus Environment


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A novel tumor pretargeting system based on complementary L-configured oligonucleotides Maik Schubert,1,# Ralf Bergmann,1,# Christian Förster,1 Wiebke Sihver,1,* Stefan Vonhoff,2 Sven Klussmann,2 Lucas Bethge,2 Martin Walther,1 Jörn Schlesinger,1 Jens Pietzsch,1,3 Jörg Steinbach,1,3 Hans-Jürgen Pietzsch1 1

Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research,

Bautzner Landstrasse 400, 01328 Dresden, Germany 2

NOXXON Pharma AG, 10589 Berlin, Germany

3

Technische Universität Dresden, School of Science, Department of Chemistry and Food

Chemistry, 01062 Dresden, Germany ‫٭‬Corresponding author at Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, POB 51 01 19, 01314 Dresden, Germany. Tel.: +49 351 260 24 24; fax: +49 351 260 32 32. E-mail: [email protected] #

These authors have contributed equally to the work.

Keywords: tumor pretargeting, mirror-image L-configured oligonucleotide, epidermal growth factor receptor (EGFR), cetuximab (C255), Cu-64, PET imaging, radioimmunotherapy

1 ACS Paragon Plus Environment

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ABSTRACT Unnatural mirror image L-configured oligonucleotides (L-ONs) are a convenient substance class for the application as complementary in vivo recognition system between a tumor specific antibody and a smaller radiolabeled effector molecule in pretargeting approaches. The high hybridization velocity and defined melting conditions are excellent preconditions of the L-ON based methodology. Their high metabolic stability and negligible unspecific binding to endogenous targets are superior characteristics in comparison to their D-configured analogs. In this study, a radiopharmacological evaluation of a new L-ONs based pretargeting system using the epidermal growth factor receptor (EGFR) specific antibody cetuximab (C225) as targetseeking component is presented. An optimized PEGylated 17mer-L-DNA was conjugated with p-SCN-Bn-NOTA (NOTA’) to permit radiolabeling with the radionuclide

64

Cu. C225 was

modified with the complementary 17mer-L-DNA (c-L-DNA) strand as well as with NOTA’ for radiolabeling and use for positron emission tomography (PET). Two C225 conjugates were coupled with 1.5 and 5.0 c-L-DNA molecules, respectively. In vitro characterization was done with respect to hybridization studies, competition and saturation binding assays in EGFR expressing squamous cell carcinoma cell lines A431 and FaDu. The modified C225 derivatives exhibited high binding affinities in the low nanomolar range to the EGFR. PET and biodistribution experiments on FaDu tumor bearing mice with directly

64

Cu-labeled NOTA’3-

C225-(c-L-DNA)1.5 conjugate revealed that a pretargeting interval of 24 h might be a good compromise between tumor accumulation, internalization, blood background, and liver uptake of the antibody. Despite internalization of the antibody in vivo pretargeting experiments showed an adequate hybridization of 64Cu-radiolabeled NOTA’-L-DNA to the tumor located antibody and a good tumor-to-muscle ratio of about 11 resulting in a clearly visible image of the tumor after 24 h up to 72 h. Furthermore, low accumulation of radioactivity in organs responsible for metabolism and excretion was determined. The presented results indicate a high potential of complementary L-ONs for the pretargeting approach which can also be applied to therapeutic radionuclides such as 177Lu, 90Y, 186Re or 188Re.



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INTRODUCTION "Learning from nature" is an wise principle for the development of a radiopharmaceutical technique introduced as tumor-pretargeting offering an alternative therapeutic approach for malignant neoplasm. As demonstrated by the natural immune system, tumor cell epitope-directed antibodies grant specific targeting with high affinity1. The large size of full antibodies cause their poor pharmacokinetics, low vascular permeability, extended retention in blood, nonspecific tissue accumulations with respect to the elimination pathways and a low tumor accumulation in solid tumors.2 Unfortunately, due to these properties, a high percentage of radiation dose is deposited not to the tumor but causing high radiation burden at critical organs. Despite, directly radiolabeled antibodies are routinely applied for tumor therapy, for example ibritumomab (Zevalin).3 The development of an effective and highly specific molecular delivery system for cytotoxic radionuclides is required to achieve therapeutic doses to antibody-pretargeted tumor cells with minimal radiation exposure to non-targeted tissue.4,5 Inspired by the efficient recognizing and fast assembling processes of complementary natural nucleic acids efforts have been made to adopt radionuclide imaging and therapy by separation of the targeting and the radioloabeling processes in vivo.6,7 Our focus is directed on the utilization of mirror image L-configured RNA- and DNA-like oligonucleotides which have been demonstrated perfect to be bioorthogonal mimics to natural D-oligonucleotides.8,9 L-configured oligonucleotides (L-ONs) derivatives show a high metabolic stability and excellent hybridization properties. In addition, they provide the ability for radiolabeling with different application-relevant radionuclides, e.g. 99m

Tc,

111

18

In and F.

10,11,12,13,14

64/67

Cu,

68

Ga,

86/90

Y,

Favorable data from radiopharmacological studies of radiolabeled

L-DNA and L-RNA derivatives include very short blood distribution phases, fast and almost exclusive renal elimination as well as very low nonspecific retention with exception of the kidneys.15,11 Studies conducted with other synthetic radiolabeled effector oligonucleotide derivatives like morpholino oligomers (MORFs) or peptide nucleic acids (PNAs) showed a rather short retention period, which might lead to incomplete hybridization due to low spatiotemporal concentration in tumor sites. Consecutive studies by our group demonstrated that PEGylation (coupling with polyethylene glycol) of a radiolabeled 17mer L-DNA effector prolonged the blood retention and reduced the accumulation of the activity in the radiationsensitive kidneys efficiently.16 The present study was performed to evaluate in vivo an epidermal growth factor receptor (EGFR)-directed tumor pretargeting system based on L-ONs and EGFR-directed antibody cetuximab (C225) as target-seeking component demonstrating the high potential of this approach. The novel pretargeting system is illustrated in Scheme 1. 3 ACS Paragon Plus Environment

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Scheme 1. A novel EGFR-directed tumor pretargeting system based on complementary Lconfigured oligonucleotides and EGFR-directed antibody C225. This report discusses the bioconjugation of a 17mer L-DNA to the epidermal growth factor epitope-specific monoclonal antibody cetuximab (Erbitux®, C225),17 the radiolabeling of the complementary 17mer-L-DNA and the C225 conjugates as well as a subsequent radiopharmacological characterization derived from in vitro and in vivo studies. Radiolabeling of the C225 derivative aimed at tracking the antibody for developmental purpose. First tumorpretargeting experiments were performed applying positron emission tomography (PET) in EGFR-bearing FaDu tumor xenografts with

64

Cu-labeled 17mer-L-DNA-10kDa-PEG-NOTA’

as best candidate versus two C225-L-DNA derivatives.

RESULTS Synthesis of tumor-specific antibody-oligonucleotide bioconjugates NOTA’3-C225-(c-LDNA)n. The antibody modified with the chelator isothiocyanate p-SCN-Bn-1,4,7-triazacyclonane-1,4,7triacetic acid (NOTA’), NOTA’3-C225-(c-L-DNA)1.5/5, was synthesized in three steps as outlined in Scheme 2. The reaction of the NOTA’ with native C225 attached an average of three NOTA’ molecules per C225 as determined by MALDI-ToF-MS (Figure S1 A, B in Supporting Information). In a second step maleimido (Mal) entities were introduced by reacting NOTA’3C225 with the heterobifunctional crosslinker 4-maleimido-butyric acid N-succinimidyl ester (GMBS). Conjugates differing in loading of Mal entities per NOTA’3-C225 were synthesized and identified by MALDI-ToF-MS (Figure S1 C, D). According to a developed one-pot



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synthesis strategy16 the disulfide protected complementary 17mer-L-DNA (c-L-DNA) was conjugated to NOTA’3-C225-Malx.

Scheme 2. Stepwise modification of antibody C225 (cetuximab) with chelator p-SCN-BnNOTA (1.), crosslinker GMBS (2.) and 17mer-c-L-DNA (3.) The number of conjugated c-L-DNA strands per antibody was quantified by UV-spectroscopy at 260 nm and 280 nm.18,19 By means of the ratios of absorbance at 260 nm and 280 nm the two different conjugates NOTA’3-C225-(c-L-DNA)1.5 and NOTA’3-C225-(c-L-DNA)5 were determined as key conjugates for further investigations. Isoelectric focusing (IEF) experiments were performed with the negatively charged antibodyoligonucleotide conjugates showing a consistent shift towards lower isoelectric point (pI) values in a range of 4.8 to 5.5 for NOTA’3-C225-(c-L-DNA)1.5 and NOTA’3-C225-(c-L-DNA)5, in comparison to unmodified C225 with pI values of 9.6 to 10.5 (Figure S2). Notably, only minor differences in pI values for NOTA’3-C225-(c-L-DNA)1.5 and NOTA’3-C225-(c-L-DNA)5 were found. Anion exchange chromatography also demonstrated the modification of C225 with c-LDNA showing multiple peaks for NOTA’3-C225-(c-L-DNA)1.5 and NOTA’3-C225-(c-L-DNA)5 (Figure S3). The product fraction of NOTA’3-C225-(c-L-DNA)1.5 was a mixture of NOTA’3C225-Mal8 (tR = 6.1 min) and derivatives with one (tR = 9.1 min), two (tR = 10.3 min) and three (tR = 11.1 min) conjugated c-L-DNA molecules (Figure S3). For NOTA’3-C225-(c-L-DNA)5 of seven peaks with retention times between 9.7 and 13.6 min were detected (Figure S3, bottom HPLC).

Synthesis of the radionuclide transporting oligonucleotide strand (the effector) The NOTA-modified and PEGylated oligonucleotide strand 1 (NOTA’-L-DNA-10kDa-PEG) was synthesized as described by Förster et al.16 A brief description of the procedures is given in the Supporting Information (Scheme S1).

Radiochemistry


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To establish preliminary experiments for in vitro pretargeting on cell lines 68Ga was used, also since it was available at any time by the generator. For

68

Ga-labeling, 2 nmol of effector

molecule 1 were reproducibly radiolabeled with 100-130 MBq of [68Ga]GaCl3 solution at room temperature obtaining [68Ga]Ga-1 in radiochemical yields (RCYs) of > 95 % and molar activities (Amols) of up to 50-60 GBq/µmol as determined by analytical radio-HPLC (Figure S4A). For

64

Cu-radiolabeling with [64Cu]CuCl2 solution, a purification step was required to

remove not complexed 64Cu. Thus, the labeling of 4.5 nmol of 1 with 180 MBq of [64Cu]CuCl2 solution resulted in a RCY of [64Cu]Cu-1 of 75% and Amols of up to 35-40 GBq/µmol determined by radio-HPLC (Figure S4B). The second part of radiochemistry performed involved 64Cu-labeling of the antibody conjugates NOTA’3-C225, NOTA’3-C225-(c-L-DNA)1.5 and NOTA’3-C225-(c-L-DNA)5. To prevent degradation of the antibody preserving binding affinities, the [64Cu]Cu2+ solution typically in 0.1 M hydrochloric acid was buffered to pH 6 before adding the solution to the antibody conjugates. The labeling was performed at 30°C with RCYs of > 95 % and Amols of 60160 GBq/µmol for ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5, ([64Cu]Cu-NOTA’)3-C225-(c-LDNA)5 and ([64Cu]Cu-NOTA’)3-C225 as determined from instant thin layer chromatography (iTLC) analysis (Figure S5). Further characterization of the radiolabeled C225 conjugates with SDS-PAGE under nonreducing conditions showed a small increase of molecular mass from ([64Cu]Cu-NOTA’)3-C225 to ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)5 (Figure S6). Under reducing conditions the antibody fragmentation into light and heavy chains is visible, for ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)5 though only in the autoradiogram (Figure S6 B). However, the bands of ([64Cu]Cu-NOTA’)3C225-(c-L-DNA)1.5 and ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)5 were blurred possibly due to the high modification with several c-L-DNA molecules on light or heavy chains causing multiple bands. These results are in agreement with data from the anion exchange chromatography analysis.

In vitro characterization of the C225 conjugates In vitro EGFR binding studies were conducted assessing the impact of chemical modification of C225 on binding affinity. In competition assays tumor cell homogenates of human epidermoid carcinoma cells (A431) and human squamous carcinoma cells of the hypopharynx (FaDu) (EGFR levels A431 2.0-2.6 × 106 EGFR/cell, FaDu 0.2-0.6 × 106 EGFR/cell)20,21,22 were incubated with different concentrations of NOTA’3-C225-(c-L-DNA)1.5, NOTA’3-C225-(c-LDNA)5 and native C225 versus ([64Cu]Cu-NOTA’)3-C225. The affinity of the C225 conjugates was both on A431 and FaDu cell homogenate still very high albeit a minor loss of affinity was ascertained compared to native C225 (Figure 1). Whereas C225 exhibited Ki values 6 ACS Paragon Plus Environment


of 0.18 ± 0.06 nM and 0.23 ± 0.07 nM for A431 and FaDu, respectively, NOTA’3-C225(c-L-DNA)1.5 and NOTA’3-C225-(c-L-DNA)5 showed Ki values of 0.46 ± 0.04 nM and 0.99 ± 0.04 nM, respectively, for A431 (Figure 1A), as well as 0.24 ± 0.06 nM and 0.73 ± 0.18 nM, respectively, for FaDu (Figure 1B). A

100

B 100 80

Binding (%)

Binding (%)

80 60 40 20

60 40 20 0

0 -11 -20

-10 -9 -8 -7 Competitor concentration (log M)

-12 -20

-11 -10 -9 -8 -7 Competitor concentration (log M)

Figure 1. Competition curves of native C225 (red circles), NOTA’3-C225-(c-L-DNA)1.5 (black circles) and NOTA’3-C225-(c-L-DNA)5 (black triangles) versus ([64Cu]Cu-NOTA’)3-C225, at (A) A431 cell homogenate and (B) FaDu cell homogenate. Results of saturation assays with ([64Cu]Cu-NOTA’)3-C225, ([64Cu]Cu-NOTA’)3-C225-(c-LDNA)1.5 and ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)5 showed dissociation constants (KDs) in a similar range within one cell line. The affinity to FaDu cells was slightly higher (Table 1). In contrast, the values for the maximal specific binding capacity (Bmax) decreased dependent on the conjugation grade (Figure 2, Table 1).

30

20

10 A 0

Bound (pmol/mg protein)

40

Bound (pmol/mg protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4 3 2 1 B 0

0

10 20 30 40 50 Competitor concentration (log M)

0

10 20 30 40 50 Competitor concentration (log M)

Figure 2. Saturation of ([64Cu]Cu-NOTA’)3-C225 (red circles), ([64Cu]Cu-NOTA’)3-C225-(c-LDNA)1.5 (black circles) and ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)5 (black triangles) binding at (A) A431 and (B) FaDu cells.


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Table 1. Data from in vitro saturation binding studies of the radiolabeled C225 derivatives to EGFR expressing cell lines A431

FaDu

KD (nM)

Bmax (pmol/mg protein)

KD (nM)

Bmax (pmol/mg protein)

4.6 ± 0.9

37.4 ± 1.9

1.3 ± 0.2

4.6 ± 0.2

([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5 3.1 ± 0.8

25.0 ± 1.5

0.8 ± 0.1

4.2 ± 0.1

([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)5 5.4 ± 0.9

21.1 ± 0.9

([64Cu]Cu-NOTA’)3-C225

1.1 ± 0.2 3.8 ± 0.1

Data are means of three samples ± SD from one experiment. The higher expression of EGFR in the A431 cells compared to FaDu cells is reflected in the 5 to 8 times higher amount of maximal binding sites Bmax.

In vitro hybridization. The melting point of the L-DNA-10kDa-PEG and the complementary cL-DNA was 78.8°C (Figure S7) assuming as rather high stability of the hybrids. Moreover, hybridization experiments using the effector (radionuclide transporter) [64Cu]Cu-NOTA’-LDNA-10kDa-PEG ([64Cu]Cu-1) and the C225 derivative NOTA’3-C225-(c-L-DNA)1.5, selected over NOTA’3-C225-(c-L-DNA)5 do to the slightly higher affinity and Bmax values,

were

performed in phosphate buffered saline (PBS) and human blood. Adding increasing concentrations of [64Cu]Cu-1 to a fixed amount of NOTA’3-C225-(c-L-DNA)1.5 the hybridization yield was estimated by agarose gel electrophoresis (Figure S8). An almost complete hybrid formation was observed at a maximum ratio of [64Cu]Cu-1 to NOTA’3-C225(c-L-DNA)1.5 of 1.53 ± 0.13 in phosphate buffer and 1.30 ± 0.11 in human blood (Figure 3). These values are in good agreement with the quantity of c-L-DNA attached to C225 as determined by UV spectroscopy (n = 1.5).


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Hybridized [64C]Cu-1/ per C225


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1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

0 5 10 15 Molar ratio [64Cu]Cu-1/NOTA3-C225-(c-L-DNA)1.5

Figure 3. Hybridization of NOTA3-C225-(c-L-DNA)1.5 with [64Cu]Cu-1 after 15 min at 37°C in phosphate buffer (circles) and human blood (triangles); graphical fitted data from autoradiographic agarose gel image (Figure S8).

In vitro pretargeting studies. In order to assess the waiting period between the administration of the tumor pretargeting components NOTA’3-C225-(c-L-DNA)1.5 and [68Ga]Ga-1, in vitro pretargeting experiments in A431 and FaDu cells were performed. For both NOTA’3-C225-(cL-DNA)5 and NOTA’3-C225-(c-L-DNA)1.5, the hybridization efficacy (bound substance per well (fmol) clearly decreased over time (Figure 4), as a result of internalization of the C225 conjugates. It was determined that ([64Cu]Cu-NOTA’)3-C225 internalized after 24 h about 50% in A431 cells and about 6% in FaDu cells (Figure S9A), whereas the binding of ([64Cu]CuNOTA’)3-C225 was both in A431 and FaDu cells similar with about 4% (Figure S9B) despite the manifold higher EGFR expression of A431 cells. Applying the higher conjugated derivative NOTA’3-C225-(c-L-DNA)5 the absolute amount of hybridization was three to four times higher relative to NOTA’3-C225-(c-L-DNA)1.5 at each time point which is in agreement with the 3.3fold higher loading of c-L-DNA per C225. Again, the higher EGFR expression of A431 compared to FaDu, since up to 10 times more hybrids are formed in A431.


600

Bound per well (fmol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B

Bound per well (fmol)

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500 400 300 200 100

60 50 40 30 20 10 0

0 0 10 20 30 40 50 60 70 Time (h)

0 10 20 30 40 50 60 70 Time (h)

Figure 4. In vitro pretargeting binding study; cells were pre-incubated with NOTA’3-C225-(cL-DNA)1.5 (circles) or NOTA’3-C225-(c-L-DNA)5 (triangles) followed by incubation with [68Ga]Ga-1 at different time points, on (A) A431 cells and (B) FaDu cells. Metabolic stability and blood clearance of [64Cu]Cu-1. In preparation of the in vivo studies, the metabolic stability of [64Cu]Cu-1 was determined in arterial blood samples of rats and resulted in intact compound of more than 90% at 120 min p.i. The blood clearance yielded an elimination half life of 15 min (Figure S10).

In vivo PET Studies with ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5. Studies on the radiopharmacological pattern of ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5 (without effector) were conducted to gain knowledge regarding blood distribution, kinetics of accumulation at tumor site, blood elimination and organ distribution in general. As shown in Figure 5 tumor accumulation increased steadily reaching a plateau about 23 h after antibody administration (SUV 1.5 ± 0.1). Until 38 h, the accumulation of the modified antibody still increased but only slightly (SUV 1.7 ± 0.10. The liver uptake at 23 h was 2.6 ± 0.4 and decreased slowly. The tumor-to-muscle ratio, which is a main parameter for background activity also reached a plateau phase 23 h post injection (p.i.) with a value of 14.6 ± 1.8. The tumor-to-liver ratio increased steadily implying tumor accumulation and concurrent liver elimination of ([64Cu]Cu-NOTA’)3C225-(c-L-DNA)1.5 over time. The elimination of ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5 from blood showed typical characteristics for large molecules, namely high accumulation in organs with strong blood circulation like lung, heart, jugular vein, liver (Figure 6).



liver

tumor

2.0

SUVmean

SUVmean

1.5 1.0 0.5 0.0 0 20

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

5 10 15 20 25 30 35 40 Time (h)

0

tumor/muscle

5 10 15 20 25 30 35 40 Time (h) tumor/liver

1.0

ratio

15 ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10

0.5

5 0

0.0 0

5 10 15 20 25 30 35 40 Time (h)

0

5 10 15 20 25 30 35 40 Time (h)

Figure 5. Representative radiopharmacokinetic curves for ([64Cu]Cu-NOTA’)3-C225-(c-LDNA)1.5 after intravenous administration (23.9 ± 0.4 MBq) into FaDu-xenografted nude mice. Representative data were calculated from dynamic PET scans. PET images after 23 h and 38 h indicate a further distribution phase characterized by a slow decrease of activity in heart, blood vessels (Figure 6) and liver (Figure 5 and 6), and an increase of activity in the tumor (Figure 5 and 6).


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Figure 6. Representative images of dynamic PET scans at different time points after

intravenously injected ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5 in FaDu xenografted NMRI nu/nu mice (A) transaxial, coronal as well as sagittal section plane, (B) maximum intensity projections coronal presented.

In vivo pretargeting PET studies. Assessing the data derived from in vitro and in vivo studies, NOTA’3-C225-(c-L-DNA)1.5 and [64Cu]Cu-1 seem to be a promising hybridization system. After injection of 4 nmol NOTA’3-C225-(c-L-DNA)1.5 24 h later about 20 MBq of [64Cu]Cu-1 (1 nmol) were administered intravenously into FaDu xenografted mice. 1 min p.i. the activity was observed in the main blood vessels and the heart (Figure 7). Already 5 min p.i. the accumulation of [64Cu]Cu-1 in the kidneys began. Due to the almost exclusively renal elimination pathway accumulated activity was also found in the bladder. A clear visualization of the tumor site was recorded at 50 min p.i. The calculated SUVPET of 0.7 ± 0.03 at this time point slightly increased to 0.9 ± 0.2 at 24 h p.i. Up to 72 h p.i. a high activity accumulation at the tumor site was measured.



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Figure 7. Representative PET images (maximum intensity projection) at different time points

after intravenous injection of [64Cu]Cu-1 in FaDu tumor bearing NMRI nu/nu mice with pretreatment of NOTA’3-C225-(c-L-DNA)1.5 24 h earlier.

In vivo pretargeting biodistribution studies. For a more detailed evaluation of the

pretargeting system biodistribution studies were conducted. Biodistribution data were gathered 24 h post [64Cu]Cu-1 injection by harvesting different organs and tissues from animals which did not receive NOTA’3-C225-(c-L-DNA)1.5 (control group, no pretargeting), and from animals that received NOTA’3-C225-(c-L-DNA)1.5 24h prior to [64Cu]Cu-1 injection (pretargeting group) (Figure 8, Table S1). Compared with the control group a significant increase of tumor accumulation could be observed 24 p.i. (SUVpretargeting 0.88 ± 0.47, SUVcontrol 0.11 ± 0.01) in the pretargeting group. The twofold higher tumor-to-muscle ratio in the pretargeting group (10.59 ± 5.02 versus 5.94 ± 0.42, respectively) demonstrates the specific hybridization of [64Cu]Cu-1 to tumor localized antibody. In analogy to the biodistribution data the pretargeting PET images (Figure 9) also show that a large amount of radiolabeled L-DNA was eliminated via the renal pathway. 24 h after injection of [64Cu]Cu-1 53.6 ± 9.4% ID for the control group compared to 79 ± 4%ID for the pretargeting group were found in the urine for elimination. However, [64Cu]Cu-1 also showed some liver accumulation.


1.4

A

control group pretargeting

1.2 1.0

SUVmean

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0.8 0.6 0.4 0.2

14

Ratio tumor/organ (SUV/SUV)

Page 15 of 32

B

control group pretargeting

12 10 8 6 4 2 0

0.0

Figure 8. (A) Biodistribution of [64Cu]Cu-1 24 h post injection in FaDu tumor bearing NMRI

nu/nu mice without (control group) and with pretreatment (pretargeting) of NOTA’3-C225(c-L-DNA)1.5 24 h earlier, (B) tumor-to-organ ratio.

Figure 9. PET-CT image of [64Cu]Cu-1 24 h post injection in FaDu tumor bearing NMRI nu/nu

mice with pretreatment (pretargeting) of NOTA’3-C225-(c-L-DNA)1.5 24 h earlier (left) and without pretreatment (control) (right). DISCUSSION

At first the monoclonal anti-EGFR antibody C225 was modified with NOTA’ to enable radiolabeling with

64

Cu and subsequent radiopharmacological studies of the final C225 14 ACS Paragon Plus Environment


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conjugates NOTA’3-C225-(c-L-DNA)1.5 and NOTA’3-C225-(c-L-DNA)5. NOTA’ has proven to be suitable for conjugation with antibodies.23,24 After covalently binding of Mal entities to NOTA’3-C225, complementary thiol-modified 17mer L-DNA strands were attached. Briefly, deprotection of disulfide protected c-L-DNA was performed under mild reaction conditions using tris-(2-carboxyethyl)phosphine (TCEP) in excess. To prevent side reactions of nonreacted TCEP with Mal entities (reduction affording succinimido groups) or with disulfide bridges of the antibody 5-azidopentanoic acid was added transforming TCEP into non-reactive TCEP-oxide and 5-aminopentanoic acid via Staudinger reaction. Two NOTA’3-C225 derivatives differing in the ratio of conjugated c-L-DNA strand per antibody molecule were synthesized. The conjugation grade for NOTA’3-C225 and NOTA’3-C225-Malx was determined by MALDI-ToF-MS. Despite intensive efforts, no reliable MALDI-ToF mass spectra could be generated for the subsequently c-L-DNA modified conjugates. However, the quantification of attached c-L-DNA was estimated by UV spectroscopy,18,19 and the radiometric hybridization with [64Cu]Cu-NOTA’-L-DNA-10kDaPEG gave consistent results for the formation of NOTA’3-C225-(c-L-DNA)1.5

and

NOTA’3-C225-(c-L-DNA)5.

The

successful

stepwise

modification of C225 was confirmed additionally by SDS-PAGE-derived isoelectric focusing showing a strongly decreased pI value after each conjugation step. Even if the pI of natural C225 has been shown to be slightly lower than in this study,25 pI values of monoclonal antibodies are in the determined range.26 A charge-dependent considerable shift towards lower pI values has been observed with conjugated mono-anions, and multiple conjugated anions,27 which is also consistent with the presented results. The retention time shifts and observation of multiple peaks in the HPLC chromatograms (Figure S3) further confirms the antibody conjugation with several c-L-DNAs. The conjugate 1 was radiolabeled with the short-lived

68

Ga (half-life 67.8 min)28 and used in

pretests for in vitro pretargeting. 68Ga was chosen since it is readily available from a generator, exposes similar binding characteristics to NOTA as

64

Cu, and does not require a purification

step after labeling. The final products of C225 conjugates showed blurred bands in SDS-PAGE autoradiograms which are typical for a heterogeneous substitution characteristics of lysine group-based protein coupling reactions.29,30 To avoid internalization issues the in vitro characterization of the C225 conjugates were performed in cell homogenate. The high affinity of the unlabeled conjugates in the cell lines A431 (0.46 and 0.99 nM) and FaDu (0.24 and 0.73 nM) compared well with unmodified C225 (~0.2 nM) that has been described before for several cell lines.31,32 A minor but still acceptable loss of affinity occurred for NOTA’3-C225-(c-L-DNA)5. Likewise, the

64

Cu-labeled C225

conjugates preserved a high affinity in both cell lines (A431 ~4.5 nM, FaDu ~1.1 nM). The conjugation with the bifunctional chelator NOTA’ has been shown to retain antibody affinity also in recent studies.24,33,34 Affinity differences of the antibody at FaDu and A431 has recently 15 ACS Paragon Plus Environment

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been suggested to arise from different interaction in the microenvironment of the EGFR, especially due to varying expressions of homo- or heterodimers of EGFR with ErB2 (HER2) levels within each cell line. Namely, cells that overexpress EGFR but have low ErbB2 expression rates (A431 cells) have exhibited lower affinity to the EGFR than cells with high ErbB2 expression and lower EGFR level (FaDu cells).35,36,37,38 However, with increasing conjugation grade, the Bmax value decreased since the potential of the conjugating groups to occupy lysine residues of the antibody close to antigen binding sites is increasing, and the steric flexibility of the conjugate to bind to EGFR is decreasing. In vitro hybridization studies of NOTA’3-C225-(c-L-DNA)1.5 with [64Cu]Cu-1 in phosphate buffer and in human blood samples successfully demonstrated the high affinity of the two binding partners of the pretargeting system. The determined binding capacity correlated very well with the calculated number of bound c-L-DNA per antibody as derived from UV spectroscopy. Investigations for in vitro pretargeting on cell lines were performed with 68Ga since the shortlived radionuclide was available at any time by a generator; moreover, both 68Ga and 64Cu form stable complexes with NOTA39,40, thus the in vitro data with 68Ga are reasonable like it would be with

64

Cu. The time dependent decrease of hybridization in the in vitro pretargeting binding

studies can be explained by internalization of the C225 conjugate. After 24h of incubation only 6% of ([64Cu]Cu-NOTA’)3-C225 was internalized in FaDu cells, but nearly 50% in A431 cells. Thus, for the pretargeting system the FaDu tumor model seemed to be more convenient, however these cells express much less EGFR. Even so, after 24 h the ([64Cu]Cu-NOTA’)3-C225 binding was similar for both cell lines. The trend for FaDu cells was still an increase of binding after 24 h. Thus, it can be inferred that in an in vivo situation FaDu tumor displays low internalization of C225 conjugates. It has been earlier reported, that labeled C225 is strong internalized in A431 cells within some hours,41,42 In FaDu cells only up to 16% of C225 internalized after 24h.43,44,45 Differences of C225 internalization rates in various cell lines could on the one hand be caused by different strong EGFR expression, but also on the different homoand hetero-dimerization of EGFR with ErB2.46 However, several tumor-pretargeting approaches have been conducted with substantial internalization depending on the cell type and antibody conjugates.47,48,49,50 For example in the pretargeting system with MORFs about 60% of a radiolabeled MORF antibody was internalized in colon cancer cells after 5 h. In spite of the strong internalization in vitro the authors showed sufficient tumor accumulation of the MORF effector in vivo.49 Likewise, in a study with a streptavidin conjugated scFv that was internalized to 60% in cultured cells after 6 h, the labeled biotin effector exhibited successful accumulation in the solid tumors of xenografted mice.47 Thus, it is valid to assume that in vitro cell assays give a merely provisory prognosis about in vivo internalization.



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For the pretargeting system elucidated in the present study the C225 conjugate with the lower conjugation grade, coupling 1.5 c-L-DNA molecules to the antibody, was selected over the conjugate with 5 c-L-DNA molecules. Even the higher conjugated C225 showed in vitro a higher hybridization grade, it displayed somewhat lower affinity and lower Bmax values. This might be an effect of steric hindrance which can be even more evident in vivo. Furthermore, repulsive forces between the phosphor lipid layer of the cell membrane and the negative phosphate backbone of the oligonucleotides could contribute to a lower binding, and consequently also to a lower tumor uptake. In PET studies with ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5 tumor accumulation was seen after 24 h and still increased until 38 h. However, liver accumulation was still high as it has been described in many studies before.51 Pharmacokinetics, recorded as PET images showed similar 64

Cu-labeled DOTA-C225-conjugate distribution in A431-tumor bearing mice,52 but also in

other tumor models53,54 showing that the c-L-DNA modification did not fundamentally change the trend. One concern for pretargeting application is the accumulation in the organs responsible for metabolism and excretion, in particular liver and kidney. Concluding, for the reported in vivo pretargeting studies a time of 24 h between injection of unlabeled NOTA’3-C225(c-L-DNA)1.5 and the complementary [64Cu]Cu-1was chosen assuming this to be an optimal balance between tumor accumulation, internalization and retention of the antibody in the bloodstream in order to minimize hybridization of radiolabeled L-DNA with circulating antibody. Before pretargeting studies were performed it was shown that the metabolic stability of the effector [64Cu]Cu-1 was very high in arterial blood samples of rats. The elimination half life of 15 min indicates a fast excretion of [64Cu]Cu-1 that can be confirmed by the low blood SUV of blood from the mice biodistribution. In the pretargeting study, after the injection of [64Cu]Cu-1 initial PET images showed organs with high blood circulation which might be explained by incomplete blood elimination of NOTA’3-C225-(c-L-DNA)1.5 and thus hybrid formation with radiolabeled L-DNA. Since radiolabeled L-ONs are almost exclusively excreted be the renal elimination pathway,11,55 a notable amount of activity was found in the kidneys, intestines and bladder. For the reduction of the blood pool activity and enhancement of tumor accumulation a longer waiting period for antibody circulation could be considered in general, but with regard to the internalization characteristics of the C225-EGFR complex no substantial improvement of tumor accumulation and tumor to background ratio might be expected in case of the C225 antibody use as model. However, the accumulation of activity in the liver was low for the pretargeting system compared with the labeled antibody. Similar low liver accumulation was reported applying direct iodine labeled C225 in A431-tumor bearing mice,56,57 however that studies resulted in lower tumor-to-muscle ratios than presented in this study. In vivo studies with the radiolabeled effector only (control group) showed significant lower background, 17 ACS Paragon Plus Environment

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namely differences in blood, liver and tumor accumulation both in biodistribution and PET scans, thus proving the functioning of the pretargeting system. Furthermore, comparing the PET data from tumor mice with the labeled antibody and with the pretargeting system a clear improvement of the tumor-to-liver ratio was demonstrated (0.6 and 1.6, respectively). Since antibody accumulation in muscle tissue is usually very low, such data will be recognized as background, and tumor-to-background ratios are generated. In this study the tumor-to-muscle ratio war slightly lower for the pretargeting system (about 11) compared with the data from 64

Cu-labeled C225 (about 14). Numerous of studies with C225 conjugates labeled with different

radionuclides showed mainly similar tumor-to-muscle ratios.51

CONCLUSION

In the present study, a further development and first in vivo evaluation of a new L-ON based pretargeting system was presented in order to overcome problems with directly labeled antibodies, i.e. to prevent radiation dose to vascularity and other healthy tissue to minimize radiotoxic, in particular, bone marrow effects2 caused by long blood residence time and low tumor accumulation. A successful methodology was established to conjugate C225 molecules with different numbers of L-ON molecules by simultaneous preservation of the affinity to the EGFR. This protocol should be able to be adapt for other antibodies as well. Secondly, it could be demonstrated that PEGylated L-DNA hybridized in vitro as well as in vivo fast with high effectiveness and specificity to the antibody-conjugated complementary oligonucleotides. These results might be an important key issue for successful treatment of cancer with pretargeting technology. Single administration of radiolabeled antibody c-L-DNA conjugate showed the typical pharmacokinetic profile of large molecules. The time period between antibody and radiolabeled L-DNA application fixed at 24 h appeared to be a good compromise between blood elimination, liver as well as tumor accumulation and internalization of the antibody-receptor complex. For optimal hybridization of the radiolabeled effector to the tumor-bound antibody conjugate it is necessary that the antibody is located at extracellular binding sites. Otherwise, the radiolabeled molecule will not find the counterpart and accumulates in a nonspecific manner, even in the tumor, which then reflects the enhanced permeability and retention effect (EPR).58 Not only the internalization itself is decisive but also the internalization rate. The latter decides if an antibody might be useful as targeting vector for pretargeted radioimmunotherapy. In the case of C225 the internalization rate of the receptor-antibody complex is notable and might explain the moderate tumor accumulation. The characteristics are comparable to the pretargeted CD22 antigen which also showed a strong tendency for internalization upon antigen binding.59,60,50 To circumvent this problem antigen-antibody systems possibly with negligible rate of internalization should be developed for future studies. Then, the pretargeting system



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might work very good also with a

68

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Ga-labeled effector for diagnostic applications. Recently,

11

even an C-labeled effector has successfully been applied in small animal PET.61 Nevertheless, based on our studies L-ONs confirmed their extraordinary high potential for the use as in vivo recognition system in pretargeting approaches. Certainly it is proposed that this method will become a versatile diagnostic and/or therapeutic tool in clinics with a broad range of different applications.

EXPERIMANTAL SECTION General. All chemicals and solvents were purchased from commercial suppliers, delivered at

analytical grade or better and used without further purification. All buffers were prepared using distilled deionized water (18.2 MΩ). L-DNA [sequence: HO-C6H12-S-S-C6H12-5’GCG GCT GTG CGG TGC GG-3’-aminohexyl-linker-10kDa-PEG] and 17mer-c-L-DNA [sequence: HOC6H12-S-S-C6H12-CCG CAC CGC ACA GCC GC -3’] were obtained from NOXXON Pharma AG Berlin (Germany). Erbitux® (C225) solution was bought from Merck. p-SCN-BnNOTA (NOTA’) was purchased from Macrocyclics (USA). The radionuclide 68Ga was eluted as [68Ga]GaCl3 from a generator of the company iThemba LABS (South Africa) in 0.6-0.8 M HCl. 64

Cu was produced at the Helmholtz-Zentrum Dresden-Rossendorf by

64

Ni(p, n)64Cu nuclear

reaction at a cyclotron (Cyclone 18/9).62 After purification 64Cu was obtained as [64Cu]CuCl2 in 0.1 M HCl. 1

H-NMR spectra were recorded at room temperature on a Varian Inova 400 spectrometer. The

analytes were dissolved in D2O using tetramethylsilane (TMS) as internal standard. Electrospray ionization mass spectrometry (ESI-MS) spectra were obtained on a Micromass Tandem Quadropole mass spectrometer (Waters Corporation, USA). The matrix-assisted laser desorption/ionization time of flight mass spectrometry (Maldi-Tof) mass spectra were measured on a Daltonic Autoflex II TOF/TOF (Bruker Daltonik). The experiments were performed in linear (LP) mode with positive polarity. For measuring of L-DNA derivatives 3hydroxypicolinic acid (3-HPA) was used as matrix. The matrix solution was a mixture of 3HPA (50 mg/ml in water/acetonitrile (MeCN) 1:1) and water/diammonium hydrogen citrate (100 mg/ml) 10:1. C225 conjugates were measured with sinapinic acid (SA, 0.1 M in MeCN/water 1:1 and 0.1% trifluoroacetic acid (TFA)). HPLC analyses were performed on a Knauer system with quaternary pump (Smartline 1000) equipped with an UV/Vis-detector (Smartline PDA 2800). Radio-HPLC chromatograms were recorded on a Knauer system with quaternary pump (Smartline 1000) equipped with a radio-detector (RAMONA, Raytest) and an UV/Vis-detector (Smartline 2500) or on a Hewlett Packard system (series 1100) equipped with a radio-detector (RAMONA, Raytest). HPLC purifications were performed on a Knauer system with a quaternary pump (WellChrom K-1001) equipped with an UV/Vis-detector (differential refractometer 298.00). The HPLC 19 ACS Paragon Plus Environment

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columns were used as follows: (A) Eurospher 100 C18 4 × 250 mm, 5 µm; (B) Eurospher 100 C18 8 × 250 mm, 5 µm; (C) X BridgeTM OST C18 4.6 × 50 mm, 2.5 µm; (D) YMC-BioPro QA-F 4.6 × 100 mm, 5 µm; (E) Zorbax C18 300SB 9.4 × 250 mm, 4 µm. System A(I): linear gradient of A (water/methanol 9:1 and 0.1Vol% acetic acid) and B (water/methanol 1:9 and 0.1Vol% acetic acid) t = 0 min, 0% B; t = 20 min, 100% B; flow 1 ml/min. System B(I): linear gradient of A (water/methanol 9:1 and 0.1Vol% acetic acid) and B (water/methanol 1:9 and 0.1Vol% acetic acid) t = 0 min, 0% B; t = 30 min, 100% B; flow 3 ml/min. System C(I): linear gradient of A (50 mM triethylammonium acetate (TEAA) pH = 6.3) and B (70% MeCN in 50 mM TEAA pH = 6.3) t = 0 min, 0% B; t = 40 min, 100% B; flow 1.4 ml/min. System C(II): linear gradient of A (50 mM TEAA pH = 6.3) and B (MeCN) t = 0 min, 0% B; t = 20 min, 100% B; flow 1.4 ml/min. System D(I): linear gradient of A (20 mM Tris/HCl pH = 8,5) and B (20 mM Tris/HCl pH = 8,5 containing 1 M NaCl) t = 0 min, 0% B; t = 20 min, 100% B; flow 1.0 ml/min. System E(I): linear gradient of A (50 mM TEAA pH = 6.45) and B (MeCN) t = 0 min, 5% B; t = 15 min, 50% B; t = 16 min, 95% B; t = 20 min, 95% B; flow 2 ml/min; column temperature 30°C. Radio TLC was performed on iTLC-SA plates (Agilent Technologies) with 0.9% NaCl solution as mobile phase. The radioactive compounds were detected with the TLC scanner (RITA, Raytest). Concentrations of UV active molecules were determined by measuring UVabsorbance at 260 nm (L-DNA and c-L-DNA derivatives) as well as at 280 nm (C225 derivatives) using a Specord 210 from Analytik Jena AG. The extinction coefficient of C225 was determined via UV calibration curve and linear regression analysis: ε280nm = 217 ± 14 µl·nmol-1·cm-1. For L-DNA and c-L-DNA sequences the extinction coefficients were calculated with oligo analyzer tool (http://eu.idtdna.com/calc/analyzer): ε260nm -1

-1

153.7 µl·nmol ·cm ,

ε260nm

c-L-DNA

=

-1

-1

147.8 µl·nmol ·cm .

Sodium

dodecyl

L-DNA

=

sulfate

polyacrylamide gel electrophoresis (SDS-Page) was performed in a vertical electrophoresis chamber from BIO-RAD (Mini-Protean® 3 cell). Proteins were separated with a 6% polyacrylamide gel under reducing and non reducing conditions (150 V, 20-30 mA, 40 min). As molecular marker it was used the HiMarkTM Pre-Stained High Molecular Weight Protein Standard (Life Technologies, USA). Gels were stained with Coomassie Brilliant Blue G250 (Thermo Fisher Scientific). Radioactivity was measured via autoradiography in a Fujix BAS 5000 image reader. Agarose gel electrophoresis was carried out in a horizontal electrophoresis chamber from Biometra® under usage of 2% agarose gels (120 V, 150-200 mA, 90 min). As molecular marker the 1 kB Plus DNA Ladder (Life Technologies, USA) was used. Gels were stained with ethidium bromide solution and monitored with a commercial available digital camera at 254 nm. Radioactivity was measured as autoradiogram in a Fujix BAS 5000 image reader. IEF was performed using a vertical electrophoresis chamber (series XCell SureLockTM,



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Life Technologies, USA) on a polyacrylamide gel pH 3-10, according to the manufacturer’s instructions. The gels were stained with Coomassie Brilliant Blue G250. Chemistry. The synthesis and characterization of NOTA’-modified and PEGylated

oligonucleotide strand 1 (NOTA’-L-DNA-10kDa-PEG) is described in the Supporting Information.

1. NOTA’3-C225. 8 ml C225 stock solution (5 mg/ml, 40 mg, 263.2 nmol) was buffer exchanged using centrifugal filter units with 30 kDa molecular weight cutoff (Pall Corporation, USA) and 50 mM NaHCO3 buffer (pH = 6.4) containing 0.9% NaCl. In the next step C225-solution was filled up with 0.5 M HEPES buffer (pH = 7.2) to a volume of 6890 µl. 27.3 mg of p-SCN-BnNOTA × 3 HCl (48.7 µmol) dissolved in 2000 µl 0.5 M HEPES buffer (pH = 7.2) was added dropwise to the antibody solution (molar ratio NOTA-derivative/C225 185:1). After a reaction time of 22 h at room temperature without stirring the modified antibody was purified using centrifugal filter units with 30 kDa molecular weight cutoff and 50 mM NaHCO3 buffer (pH = 6.4) containing 0.9% NaCl. The recovery of NOTA3-C225 was almost quantitatively (260 nmol, 99% yield determined by UV/Vis measurement). HPLC D(I): tR = 5.1 min. MS (Maldi-TOF+): m/z = 155000. IEF: pI = 7.8 – 8.4.

2. General procedure for the synthesis of maleimide fuctionalized NOTA’3-C225-Malk derivatives. To a solution of NOTA’3-C225 (100.5 nmol) in 50 mM NaHCO3 buffer (pH = 6.4, containing 0.9% NaCl) was added phosphate buffer (pH = 7.0, containing 0.9% NaCl) until a volume of 3350 µl. In the next step 4-maleimidobutyric acid N-hydroxy-succinimide ester dissolved in 50 µl DMSO was added to the antibody solution. For the synthesis of the low modified antibody conjugate NOTA’3-C225-Mal8 0.28 mg (1005 nmol) and for the high modified conjugate 1.41 mg (5025 nmol) were used. After a reaction time of 5 h at 22°C without stirring the solution was purified using centrifugal filter units with 30 kDa molecular weight cutoff and phosphate buffer (pH = 6.0) containing 0.9% NaCl. For both conjugates the recovery was almost quantitatively (100.5 nmol, 99% yield determined by UV/Vis measurement).

NOTA’3-C225-Mal8:

HPLC

D(I):

tR = 5.9

min.

MS

(Maldi-TOF+):

m/z = 156460. IEF: pI = 5.9 – 7.3. NOTA3’-C225-Mal33: HPLC D(I): tR = 5.7 min. MS (MaldiTOF+): m/z = 160120. IEF: pI = 4.6 – 5.2.

3. General procedure for the synthesis of maleimide fuctionalized NOTA’3-C225-(c-L-DNA)n derivatives. An aliquot of NOTA’3-C225-Malk solution (100 nmol in ≈ 2 ml phosphate buffer pH = 6 containing 0.9% NaCl) was mixed with an amount of 17mer-c-L-DNA that was deprotected according to the protocol described in supplemental data. The final volume of the reaction mixture ranged between 4 and 4.5 ml. For the synthesis of the low modified antibody conjugate NOTA’3-C225-(c-L-DNA)1.5 starting from NOTA’3-C225-Mal8 a 10 fold excess and for the synthesis of the high modified antibody conjugate NOTA’3-C225-(c-L-DNA)5 starting


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from NOTA’3-C225-Mal33 a 20 fold excess of 17mer-c-L-DNA was used. The reaction mixture was incubated at room temperature over night without stirring. In the next step the solution was purified using centrifugal filter units with 30 kDa molecular weight cutoff and phosphate buffer (pH = 6.0) containing 0.9% NaCl. To remove nonspecifically bound 17mer-c-L-DNA from the antibody surface the resulting antibody solution was incubated at room temperature over night in 0.5 M phosphate buffer (pH = 6.0, containing 2 M NaCl). Subsequently the antibody conjugates were buffer exchanged using centrifugal filter units with 30 kDa molecular weight cutoff and 50 mM ammonium acetate buffer (pH 6.0, containing 0.9% NaCl). The number of bound oligonucleotides per C225 was quantified with spectrophotometrical methods.18,19 This required different absorption maxima of the respective antibody (λmax C225

= 280 nm) and the conjugated oligonucleotide (λmax

c-L-DNA

= 260 nm). Assuming that the

conjugation with c-L-DNA does not affect the extinction coefficients of the individual compounds Lambert Beers equations were formulated at 260 nm and 280 nm. The extinction coefficients were determined via UV/Vis calibration curves and linear regression analysis: εC225, 280nm =

217 ± 14 µl/nmol·cm,

εC225,

260nm =

97 ± 6 µl/nmol·cm,

εc-L-DNA, 280nm = 112

± 5 µl/nmol·cm and εc-L-DNA, 260nm = 148 ± 7 µl/nmol·cm. Based on the 260 nm/280 nm absorbance ratio the conjugation degree was calculated with following equation: Equation 1

=

× − × × − ×

NOTA’3-C225-(c-L-DNA)1.5: Yield 88%. HPLC method 6: tR1: 6.1 min, tR2: 9.1 min, tR3: 10.3 min, tR3: 11.1 min. IEF: pIband1 = 5.2 - 7.5, pIband2 = 6.2 - 6.7. NOTA’3-C225-(c-L-DNA)5: Yield 90%. HPLC method 6: tR: 9.7 - 13.6 min,: pI = 4.8 - 5.3 Radiochemistry. 68Ga labeling of NOTA’-L-DNA-10kDa-PEG 1. To a solution of 1 (2 nmol) in

1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH = 7.0) 100-130 MBq of [68Ga]GaCl3 solution (50-100µL) was added. To adjust the pH to about 5 the volume of MES buffer was calculated with following formula: Vbuffer = Veluate × 1.065. The reaction mixture was incubated for 35 min at 30°C and shaken at 600 min-1 in a thermomixer. Thereafter, an equivalent

of

the

labeling

solution

(1.5 µl)

was

added

to

40 µl

of

1.9 µM

ethylenediaminetetraacetic acid (EDTA) solution in 0.1 M MES buffer (pH = 6.0) to complex unreacted [68Ga]Ga3+ as ([68Ga]Ga-EDTA)-. After a reaction time of 10 min at room temperature the RCY and radiochemical purity (RCP) of [68Ga]Ga-1 were determined by HPLC. [68Ga]Ga-1: HPLC C(II): tR = 9.3 min. RCY ≥ 99%. RCP ≥ 99% . Amol = 54 GBq/µmol. 64

Cu labeling of NOTA’-L-DNA-10kDa-PEG 1. To a solution of 1 (4.5 nmol) in 240 µl 0.1 M

MES buffer (pH = 6.0) 180 MBq [64Cu]CuCl2 solution (50-100 µL) was added. Subsequently the reaction mixture was incubated in a thermomixer at 30°C and 600 min-1. After a reaction 22 ACS Paragon Plus Environment


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time of 35 min 6 µl of 0.75 mM EDTA solution was added to complex unreacted [64Cu]Cu2+ as ([64Cu]Cu-EDTA)2- and monitored by HPLC. In the next step the radiolabeling mixture was purified by centrifugal filtration (14500 min-1, 10 min, recovery of activity 70-80%) and rebuffered with phosphate buffer (pH = 7.0). [64Cu]Cu-1: HPLC C(II): tR = 9.3 min. RCY = 73.4%. RCP ≥ 99% . Amol = 36.7 GBq/µmol.

General procedure for 64Cu-labeling of NOTA’3-C225 and NOTA’3-C225-(c-L-DNA)n. 3 nmol of antibody stock solution were diluted in 200 µl 1 M MES-buffer (pH = 6.0). An equivalent of about 50 MBq of [64Cu]CuCl2-solution in 0.01 M HCl was mixed 1:1 with 1 M MES-buffer (pH = 6.0) to adjust the pH to 6.0 and then added to the antibody solution. The reaction mixture was incubated at 30°C for 35 min in a thermomixer and the vial was swirled occasionally by manual stimulation. Subsequently, 4 µl of an 0.75 mM EDTA stock solution was added to the reaction to bind unreacted [64Cu]Cu2+ as ([64Cu]Cu-EDTA)2-. After a reaction time of 10 min at room temperature the radiolabeling mixture was monitored by radio-iTLC for complete complexation of [64Cu]Cu2+. ([64Cu]Cu-NOTA’)3-C225 TLC: Rf = 0. RCY ≥ 98%. RCP ≥ 98% . Amol = 61.0 GBq/µmol. ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5 TLC: Rf = 0. RCY ≥ 98%. RCP ≥ 98% . Amol = 58.1 GBq/µmol. ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)5 TLC: Rf = 0. RCY ≥ 98%. RCP ≥ 98% . Amol = 55.6 GBq/µmol. In vitro hybridization. To evaluate the hybridization stability the melting point of the L-DNA-

10kDa-PEG and the complementary c-L-DNA was determined (Supporting Information). Furthermore, the stability on buffer and blood was investigated. To 0.02 nmol of NOTA’3C225-(c-L-DNA)1.5 in 50 µl phosphate buffer (pH 7.0) or human blood aliquots of [64Cu]Cu-1 in different molar ratios of NOTA’3-C225-(c-L-DNA)1.5/[64Cu]Cu-1 from 1:0.1 to 1:15 were added to the antibody solutions. After a reaction time of 15 min at 37°C and shaking with 300 min-1 the aliquots were analyzed by agarose gel electrophoresis. From samples containing human blood cellular fractions were removed by centrifugation (5 min, 2000 min-1). Cell culture. The experiments were performed with the EGFR expressing cell lines A431

(epidermoid carcinoma) and FaDu (squamous pharyngeal carcinoma). A431 cells were cultivated in DMEM with 10% fetal calf serum (FCS), FaDu cells in RPMI medium with 10% FCS, respectively, in an incubator with humid atmosphere of 95% air and 5% CO2 at 37°C. The media contained 1% penicillin-streptomycin. Competition assay. To avoid ligand-receptor internalization measuring only receptor binding

cell homogenate was used. After washing the cells 3 x with PBS and scraping them in 5 mL of medium they were homogenized on ice by 20 strokes with pestle and glass tube (PotterElvehjem). After centrifugation for 5 min at 600 g and re-suspension in medium the cell homogenate was incubated with varying concentrations of NOTA’3-C225-(c-L-DNA)1.5, NOTA’3-C225-(c-L-DNA)5 and C225 (0.8 pM - 5 µM), and the in-house established radio23 ACS Paragon Plus Environment

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standard ([64Cu]Cu-NOTA’)3-C225 (0.5 nM). After 1 h the incubations were stopped by filtration through glass microfiber filters (GF/B), presoaked with 0.3 %polyethylenimin, using a cell harvester (Brandel). Unbound radiostandard was removed by washing the filters 4 x with ice-cold PBS. Punched filter units were transferred into vials and counted in a gamma counter (Wizard, Perkin Elmer). In vitro saturation assays. About 100,000 A431 respective 40,000 FaDu cells were plated in

each well of 24-well plates. A confluency of 80-90% was achieved after one day for A431 and after four days for FaDu cells. The cells were incubated with different concentrations of ([64Cu]Cu-NOTA’)3-C225, ([64Cu]Cu-NOTA’)3-C225-(c-L-DNA)1.5 or ([64Cu]Cu-NOTA’)3C225-(c-L-DNA)5 (0.01 nM to 50 nM) for 1 h in an incubator. Adjacent cell samples received additionally 5 µM C225 to obtain the nonspecific binding. After washing the cells 3 x with PBS they were lysed with 0.5 ml/well 1% SDS in 0.1 M NaOH. 30 min later after intense shaking the impulse rate of the cell fractions were measured in a gamma counter. In vitro pretargeting binding study. Cells were prepared as for saturation assays. 1 or 3 days

later NOTA’3-C225-(c-L-DNA)1.5 or NOTA’3-C225-(c-L-DNA)5 was diluted in medium to a 4 nM solution for FaDu cells or a 40 nM solution for A431cells. The cell culture medium was replaced with 0.5 ml/well of the prepared antibody solutions, and the cells were incubated for 1h. Subsequently, the antibody solutions were aspirated, cells washed 3 x with PBS and 0.5 ml/well fresh medium was added. At measurement time points of 1 h, 4 h, 24 h, 48 h and 72 h after the antibody treatment the medium was replaced from the cells with 0.5 ml/well of a stock solution with 20 nM of [68Ga]Ga-1 for FaDu cells or 40 nM of [68Ga]Ga-1 for A431 cells. After an incubation time of 5 min on a thermal shaker at 37°C [68Ga]Ga-1 solutions were removed from the cell culture plates, the cells washed 3 x with 0.5 ml/well PBS and finally lysed with 0.5 ml/well 1% SDS in 0.1 M NaOH. After 30 min of intense shaking the impulse rate of the cell fractions was measured in a gamma counter. Internalization. The internalization of ([64Cu]Cu-NOTA’)3-C225 was performed essentially as

described by Schaffar et al.63, however with modifications. A431 or FaDu cells were cultured in 48-well plates (5 x 104 cells/well) over night. Then, the cells were incubated with 4 nM ([64Cu]Cu-NOTA’)3-C225 (~ 40 kBq/well) for 15 min, 3 h and 24 h in an incubator. Adjacent samples received 0.64 µM C225 to obtain the nonspecific part. The incubation was finished by 2 x washing with cold PBS and adding 450 µL acid wash buffer (0.1 M glycine, 0.15 M NaCl at pH 3 with HCl). After 10 min the buffer was transferred into vials to measure the activity (membrane bound ([64Cu]Cu-NOTA’)3-C225). The cells in the wells were lysed with 0.1 M NaOH + 1% SDS, transferred into vials and also measured in a gamma counter.



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The protein amount in saturation, pretargeting and internalization assays was quantified with bicinchoninic acid protein assay (Pierce, Thermo Scientific). The data points of the hybridization, competition, saturation and pretargeting binding assays were analyzed by a nonlinear curve fitting program (GraphPad Prism 5.02). Animals and tumor models. The animal studies were approved from the local animal research

committee at the Landesdirektion Dresden according to institutional guidelines and the German Animal Welfare Regulations. Rats (Wistar Unilever, HsdCpb: Wu, Harlan Winkelmann GmbH, Borchen, Germany) and mice (NMRI, nu/nu) were housed under standard conditions with free excess to food and tap water. 2 x 106 FaDu cells were transplanted subcutaneously in the right thigh of anesthetized (8% desfluran in 30% oxygen/air mixture) mice. The experiments were carried out when the tumors reached a size of 8-13 mm. Metabolic stability of [64Cu]Cu-1. 60 MBq [64Cu]Cu-1 (1.5 nmol) was injected intravenously

into the tail vein of male with desfluran anesthetized rats. A catheder was set into the right femoral artery to take arterial blood samples 1, 5, 10, 20, 30, 60 and 120 min after radiotracer injection. The blood samples were centrifuged immediately to remove erythrocyte fraction. Before blood plasma samples were analyzed for [64Cu]Cu-1 content by HPLC system (Hewlett Packard Series 1100, radiodetector: Raytest Ramona, column: Zorbax C18 300SB 9.4 x250mm 4µm, eluates: A = triethylamine 50 mM pH 6.45, 0.1% TFA, B = MeCN, 0.1% TFA; gradient: during 15 min from 5 to 50% B; 1min to 95% B; 4 min 95% B; during 1 min to 5% B; 9 min 5% B) containing proteins were precipitated with 60% MeCN solution. The resulting precipitate was removed as pellets after centrifugation. Small animal PET studies. PET studies were performed in FaDu tumor bearing mice under

desfluran anesthesia. Ca. 20 MBq of the direct labeled antibody ([64Cu]Cu-NOTA’)3-C225-(cL-DNA)1.5 was injected into the tail vein of 4 animals. For pretargeting experiments about 20 MBq [64Cu]Cu-1 (~1 nmol) was injected 24 h after single administration of 2.9 nmol NOTA’3C225-(c-L-DNA)1.5. PET scans were performed at a Concord MicroPET® P4 (CTI Molecular Imaging, Siemens Medical Solutions). The images were generated with Rover software (ABX GmbH, Radeberg, Germany). For demonstration of the time course, the data points were fitted by a nonlinear curve fitting program (GraphPad Prism 5.02). Biodistribution study. For a pretargeting biodistribution study altogether 11 FaDu tumor

bearing NMRI nu/nu mice with a body weight of 30.9 ± 1.5 g were used. 9 mice thereof received a single dose of 2.9 nmol NOTA’3-C225-(c-L-DNA)1.5 and 24 h thereafter simultaneously with 2 control mice a dose of 17.6 ± 2.3 MBq [64Cu]Cu-1 (~ 1nmol) into the tail vein. During the injections the animals were anesthetized with desfluran. Another 24 h later the animals were sacrificed under desfluran by cardiocentesis. Organs and tissues of interest were


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removed and weighed, and the radioactivity was quantified using a gamma counter. The measured activity values were related to the amount of injected activity. Standardized uptake values (SUV) were calculated using the following equation:

SUV =

tissue activity× body weight injected activity× tissue weight

ACKNOWLEDGEMENT

The authors thank Ms. Ulrike Gesche, Ms. Regina Herrlich and Ms. Andrea Suhr for their excellent technical assistance. The authors further wish to thank the staff of the Production of Radiopharmaceuticals Department at the Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf for production of 64Cu.

Supporting Information Available

ABBREVIATION

Amol, molar activity; Bmax, maximum binding capacity; C225, cetuximab; EDTA, ethylenediaminetetraacetic acid; EGFR, epidermal growth factor receptor; ErB2 (HER2), epidermal growth factor receptor type 2; ESI-MS, electrospray ionization mass spectrometry; FCS, fetal calf serum; GMBS, 4-maleimido-butyric acid N-succinimidyl ester; IEF, isoelectric focusing; iTLC, instant thin layer chromatography; KD, equilibrium dissociation constant; Ki, equilibrium inhibition constant; L-ONs, L-configured oligonucleotides; Mal, maleimido; MALDI-ToF-MS, matrix-assisted laser desorption/ionization - time of flight mass spectrometry; MeCN, acetonitrile; MES, 2-(N-morpholino)ethanesulfonic acid; MORF, morpholino oligomer; NOTA, 1,4,7-triazacyclonane-1,4,7-triacetic acid; NOTA’, isothiocyanate p-SCN-Bn-NOTA; PBS, phosphate buffered saline; PEG, polyethylene glycol; PET, positron emission tomography; PNA, peptide nucleic acid; RCP, radiochemical purity; RCY, radiochemical yield; SA, sinapinic acid; SDS-Page, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SUV, standardized uptake value; TCEP, tris-(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; tR, retention time



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