Pretargeted PET Imaging Using a Bioorthogonal 18F-Labeled trans

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Communication 18

Pretargeted PET imaging using a bioorthogonal Flabeled trans-cyclooctene in an ovarian carcinoma model Emilie Billaud, Sarah Belderbos, frederik Cleeren, Wim Maes, Marlies Van de Wouwer, Michel Koole, Alfons Verbruggen, Uwe Himmelreich, Nick Geukens, and Guy Bormans Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00635 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 1, 2017

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Pretargeted PET imaging using a bioorthogonal

18

F-labeled trans-cyclooctene in an

ovarian carcinoma model

Emilie M. F. Billaud,‡ Sarah Belderbos,§ Frederik Cleeren,‡ Wim Maes,† Marlies Van de Wouwer,† Michel Koole,∥ Alfons Verbruggen,‡ Uwe Himmelreich,§ Nick Geukens,† and Guy Bormans*,‡

‡

Laboratory for Radiopharmaceutical Research, Department of Pharmaceutical and Pharmacological

Sciences, KU Leuven, Campus Gasthuisberg, O&N2, Herestraat 49, Box 821, 3000 Leuven, Belgium §

Biomedical MRI/MoSAIC, Department of Imaging and Pathology, KU Leuven, Campus Gasthuisberg,

O&N1, Herestraat 49, Box 505, 3000 Leuven, Belgium †

PharmAbs, the KU Leuven Antibody Center, KU Leuven, Campus Gasthuisberg, O&N2, Herestraat 49,

Box 820, 3000 Leuven, Belgium ∥

Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, University Hospital

and KU Leuven, Herestraat 49, box 7003, 3000 Leuven, Belgium * Corresponding author: [email protected]

Abstract In cancer research, pretargeted positron emission tomography (PET) imaging has emerged as an effective two-step approach that combines the excellent target affinity and selectivity of antibodies with the advantages of using short-lived radionuclides such as fluorine-18. One possible approach is based on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between tetrazines and transcyclooctene (TCO) derivatives. Here, we report the first successful use of an

18

F-labeled small TCO

compound, [18F]1 recently developed in our laboratory, to perform pretargeted immuno-PET imaging. The study was performed in an ovarian carcinoma mouse model, using a trastuzumab-tetrazine conjugate.

Communication Positron emission tomography (PET) is a highly sensitive non-invasive technology, allowing molecular imaging of diseases such as cancer.1 It is useful for diagnosis and staging of diseases, as well as ACS Paragon Plus Environment

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for selection of patients eligible to a specific treatment (personalized medicine), and for monitoring treatment response. PET produces three-dimensional images representing the distribution and concentration of a positron emitter in vivo, after intravenous injection (IV) of a radiopharmaceutical in a patient.2 The most frequently used PET radionuclide is fluorine-18, given its favorable decay properties (97% β+ emission, 634 keV maximum β+ energy), and short β+ trajectory in tissues (400 GBq per batch) with a cyclotron.3 Pretargeted PET imaging has emerged as an effective in vivo approach to combine short-lived radionuclides such as fluorine-18 with antibodies, which display excellent affinity and selectivity for tumor targets.4,5 This two-step approach overcomes the limitations associated with the slow pharmacokinetics of antibodies (multiday biological half-lives), by allowing a maximum uptake of an antibody in the tumor and a sufficient clearance from non-targeted tissues, before injection of the radioactive compound for PET imaging.6 Moreover, this pretargeting approach reduces the radiation burden for the patient compared to radioimmunoconjugates directly labeled with longer-lived radionuclides such as zirconium-89 (t1/2 = 78.4 h).7 The inverse-electron-demand Diels–Alder (IEDDA) reaction between 1,2,4,5-tetrazines and transcyclooctene (TCO) derivatives is a fast, selective, high-yield, biocompatible, and bioorthogonal click ligation,8-12 which has already proven to be suitable for in vivo pretargeted nuclear imaging using immunoconjugates.7,13-25 So far, the use of fluorine-18 for pretargeted immuno-PET based on the IEDDA reaction has been demonstrated with radiolabeled tetrazine derivatives,26,27 but not with radiolabeled small TCO compounds although some radiofluorination procedures were developed.28-31 However, small TCO compounds would have specific advantages with regard to stability and pharmacokinetics, for instance to cross the blood-brain barrier. Recently, we developed a new

18

F-labeled TCO ([18F]1, Figure 1) that demonstrated high

reactivity for IEDDA reactions, and suitable in vivo pharmacokinetics for pretargeting applications.32 This derivative was prepared via diastereoselective synthesis, and the trans-for-cis isomerization step was performed using an innovative micro-flow photochemistry process. Proof-of-principle PET imaging experiments with [18F]1, on a prostate tumor mouse model injected with a tetrazine-coupled prostatespecific membrane antigen antagonist 10 min before radiotracer injection, allowed clear visualization of the tumor tissue, due to the 18F-labeled conjugate formed by the IEDDA reaction.32 On the basis of these promising results, we aimed to perform pretargeted immuno-PET imaging using for the first time a

18

F-labeled small TCO compound, [18F]1, and a tetrazine-derivatised antibody.

Trastuzumab, a well-characterized humanized monoclonal antibody specific to the human epidermal ACS Paragon Plus Environment

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growth factor receptor 2 (HER2), was chosen as model antibody. It has been approved by the Food and Drug Administration and the European Medicines Agency for treatment of HER2-positive breast and gastric cancers. Indeed, HER2 is overexpressed in many forms of cancers such as breast (15-30%), gastric (10-30%), and ovarian (20-30%) cancers.33 Moreover, trastuzumab has also been employed for PET imaging of HER2 expression, after labeling with relatively long-lived radionuclides such as copper-64 (t1/2 = 12.7 h), iodine-124 (t1/2 = 100.3 h) or zirconium-89, since it can provide valuable diagnostic information influencing patient management.34-38 In order to successfully apply [18F]1 for pretargeted immuno-PET, stability was preferred over reactivity for the choice of tetrazine, as the tetrazine-derivatised antibody is injected several days before [18F]1. Encouraged by the results reported by Karver et al.39 and Selvaraj et al.,40 we decided to synthesize two trastuzumab-tetrazine conjugates (Figure 1), one bearing a 3-(4-(trifluoromethyl)phenyl)6-phenyl-1,2,4,5-tetrazine (2) and one bearing a 3-methyl-6-phenyl-1,2,4,5-tetrazine (3). With the aim of further increasing their hydrophilicity and their stability towards enzymatic degradation, tetrazines were derivatised with polyethylene glycol (PEG) chains.41 Here, we report (i) the syntheses of two new trastuzumab-tetrazine conjugates, 2 and 3; (ii) their in vitro stability in rat plasma; (iii) binding of the most stable immunoconjugate for HER2; (iv) cell internalization studies; (v) in vivo pretargeted immuno-PET imaging using 3 and [18F]1 in an ovarian carcinoma mouse model.

O H

F

3

O

O

H N O

18

O [18F]1

H

H N

H

O

O

3

O O

O

N H

3

trastuzumab N N

N N

2

O

N H antibody

N N

N N

3: antibody = trastuzumab 4: antibody = vedolizumab

CF3

Figure 1. Chemical structures and conceptual schematics of compounds used in this study

ACS Paragon Plus Environment


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In order to obtain immunoconjugate 2, tetrazine derivative 8 was first synthesized, as shown in Scheme 1. Briefly, amidation between carboxylic acid of compound 540 and amine of commercially available tert-butyl 12-amino-4,7,10-trioxadodecanoate, in the presence of HATU and N,Ndiisopropylethylamine, yielded PEGylated tetrazine derivative 6. After tert-butyl ester deprotection, compound 7 reacted with N-hydroxysuccinimide in the presence of N,N′-dicyclohexylcarbodiimide and 4(dimethylamino)pyridine, leading to activated ester derivative 8. The latter or commercially available 2,5dioxopyrrolidin-1-yl 1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)-3,6,9,12-tetraoxapentadecan-15-oate (Tz-PEG4-NHS) were then conjugated with trastuzumab via amidation on lysine residues, yielding trastuzumab-tetrazine conjugates 2 or 3, respectively. The number of tetrazine moieties attached to the antibody was determined via (i) analysis of the crude reaction mixture by size-exclusion chromatography (SEC)-HPLC at 520 nm (characteristic tetrazine absorption) and/or (ii) UV-visible spectrophotometry following the reaction of purified 2 or 3 with Cy5-TCO, a TCO-bearing fluorescent probe (see Supporting Information). An average of 0.5 and 7.7 reactive tetrazine moieties per antibody was determined for immunoconjugates 2 and 3 respectively. This difference can be due to the reaction time of the conjugation or to the physicochemical properties of the tetrazines (hydrophobicity, stability, etc). H N

O H2N

N N

O

HATU DIPEA DMF RT, 17 h

OH

H N

O

O

TFA CH2Cl2

Ot-Bu

3

O

N N

H N

O

N N

O

N N

RT, 17 h

NH

O

O

N N

O

N N

NH

Ot-Bu

O

3

CF3 H N

N N

CF3

5

H N

O

NHS DMAP, DCC 1,4-dioxane

O

N N

NH

O

O

OH

N N

CF3

NH

O O

O

O 3

CF3

quantitative

O

N N

RT, 17 h

7

7

O

3

CF3

OH 3

94%

6

O

8

N O

61%

Scheme 1. Synthesis of tetrazine derivative 8 DCC: N,N′-dicyclohexylcarbodiimide, DIPEA: N,N-diisopropylethylamine, DMAP: 4-(dimethylamino)pyridine, DMF: N,N-dimethylformamide, HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3oxid hexafluorophosphate, NHS: N-hydroxysuccinimide, TFA: trifluoroacetic acid.


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The stability of both trastuzumab-tetrazine derivatives was then assessed in rat plasma, at 37 °C. At selected time points, a sample was mixed with Cy5-TCO and analyzed by SEC-HPLC at 650 nm (characteristic Cy5 absorption) to determine the resulting reactivity. The percentage of trastuzumabtetrazine conjugate that reacted with Cy5-TCO was determined to be 27% after 5 days incubation for 2, and 55% after 7 days incubation for 3 (Figure S5). Therefore, the latter was selected for subsequent experiments and in vivo pretargeting applications. In order to assess whether the binding of immunoconjugate 3 for HER2 is retained, a flow cytometry assay was performed. For this assay, SKOV-3 human ovarian carcinoma cells, highly expressing HER2,42 were incubated either with trastuzumab or with trastuzumab-tetrazine derivative 3. The binding to HER2 was then assessed by probing with an anti-human immunoglobulin G1 tagged with fluorescent allophycocyanin for fluorescence-activated cell sorting analysis. Results demonstrated that conjugation with tetrazine did not alter trastuzumab binding to HER2 (Figure S6), since there was no significant difference between the mean fluorescence intensity after incubation with trastuzumab (30 084 ± 781 arbitrary units) or trastuzumab-tetrazine derivative 3 (31 995 ± 859 arbitrary units). Therefore, immunoconjugate 3 seems suitable for pretargeting applications in HER2-positive SKOV-3 ovarian carcinoma models. Internalization of trastuzumab-tetrazine conjugate 3 was then investigated in SKOV-3 cells. It has been previously shown that trastuzumab internalizes and recycles back to the plasma membrane of cells with high HER2 expression,43 such as SKOV-3. In such cells, trastuzumab internalization is HER2mediated, and after intracellular trafficking, both the antibody and HER2 are recycled.43 Although cell surface persistence of the antibody is not an absolute prerequisite for pretargeting applications,7 we sought to validate the behavior of trastuzumab-tetrazine derivative 3 and TCO [18F]1 in a pretargeting methodology. SKOV-3 cells were thus incubated at 37 °C with immunoconjugate 3 for 60 minutes, and then washed before incubation with [18F]1 for 15 minutes. To check the specificity for HER2, a blocking assay using trastuzumab, co-incubated with derivative 3, and a control assay using [18F]1 only (no pretargeting), were also carried out under the same conditions. To verify that the radioactive uptake is due to the IEDDA reaction, another assay was performed by incubating the cells for 10 minutes with a nonradioactive TCO, Cy5-TCO, before incubation with [18F]1. For all assays, the radioactive uptake was measured in the surface-bound fraction after acid wash with a glycine hydrochloride solution (pH 2.8) and in the internalized fraction after lysis of the cells. Results are displayed in Figure 2.



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Figure 2. Cell surface binding and internalization following incubation of HER2-positive SKOV-3 cells with immunoconjugate 3 (1 µM) for 60 min and then with [18F]1 (0.7 MBq.mL-1) for 15 min. Specificity for HER2 was evaluated by using trastuzumab (50 µM) as a blocking agent or by incubating the cells with [18F]1 only (no pretreatment with 3). Selectivity of the IEDDA reaction between trastuzumabtetrazine derivative 3 and TCO [18F]1 was verified by incubating the cells with non-radioactive Cy5-TCO (50 µM) for 10 min before the addition of [18F]1. Cells were incubated at 37°C. Data are expressed as mean ± standard error of the mean (N = 3). Significant HER2-specific binding to SKOV-3 cells was observed in the pretargeting experiment (3 + [18F]1) with a total bound fraction of 23.69 ± 1.21%, compared to 1.22 ± 0.11% (p < 0.0001) when trastuzumab was used as a blocking agent, and 0.38 ± 0.02% (p = 0.0003) following incubation of [18F]1 without pretreatment. We demonstrated that this specific binding is clearly due to the

18

F-labeled

conjugate formed by the IEDDA reaction between 3 and [18F]1, since a total bound fraction of only 0.44 ± 0.05% (p < 0.0001) was determined when non-radioactive Cy5-TCO was incubated before [18F]1. Following incubation of SKOV-3 cells with 3 and [18F]1, 86% of the total bound fraction is internalized. The radioactivity fraction in the lysate might correspond to the

18

F-labeled conjugate formed by the

IEDDA reaction at the cell surface and subsequently internalized with the receptor, and/or due to passive transport of [18F]1 through the cell membrane and subsequent intracellular IEDDA reaction. These results were confirmed by repeating the experiment (experiment 2, Figure S7). The in vivo pretargeted PET imaging approach using trastuzumab-tetrazine derivative 3 and TCO 18

[ F]1 was then evaluated in SKOV-3 tumor-bearing nude mice. To this end, immunoconjugate 3 (100 µg; 0.67 nmol) was administered IV to the mice, followed 2 to 3 days later by [18F]1 (IV; 6-12 MBq; 0.67 nmol). Whole-body dynamic (0-60 min postinjection) and static (120 min postinjection) microPET scans were acquired. Immuno-PET imaging allowed visualization of the tumor, as shown in Figures 3 and S8, with a significantly higher tumor uptake compared to muscle uptake, from 20 min to 1 h post tracer injection. Comparable results were obtained 2 and 3 days after injection of immunoconjugate 3, as shown ACS Paragon Plus Environment

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in Figure 4. In order to demonstrate the specificity of this approach for HER2, a control experiment (IV injection of [18F]1 without pretargeting pretreatment) and a non-specific pretargeting experiment were also carried out in the same tumor model. For the non-specific experiment, tetrazine-derivatised vedolizumab 4 (100 µg; 0.67 nmol; synthesis described in Supporting Information) was used, as vedolizumab does not target HER2 but the integrin α4β7.44 Results are presented in Figures 4 and S8. No significant difference in tumor and muscle uptake was found in the control and non-specific pretargeting experiments, thus proving the specificity of the approach involving trastuzumab-tetrazine derivative 3 followed by injection of [18F]1 for HER2.

Figure 3. (A) In vivo pretargeted PET imaging 2 h postinjection (IV) of bioorthogonal [18F]1 (11 MBq; 0.67 nmol), 2 days after IV injection of trastuzumab-tetrazine conjugate 3 (100 µg; 0.67 nmol) in a nude mouse bearing a subcutaneous SKOV-3 xenograft on the right shoulder. Tomographic slices bisect the tumor. The tumor is indicated by an arrow. (B) Time-activity curves from 0 to 60 min after IV injection of [18F]1 (11 MBq; 0.67 nmol), 2 (top, N = 3) or 3 days (bottom, N = 3) after IV injection of trastuzumab-tetrazine conjugate 3 (100 µg; 0.67 nmol) in SKOV-3 tumor-bearing nude mice. Data are expressed as standardized uptake value (SUV).



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Figure 4. Tumor-to-muscle activity ratios after IV injection of [18F]1 (11 MBq; 0.67 nmol), 2 or 3 days after IV injection of trastuzumab-tetrazine conjugate 3 (100 µg; 0.67 nmol) are comparable and are significantly higher than in the control experiment (IV injection of [18F]1 (6 MBq) without pretargeting pretreatment) and the non-specific experiment (IV injection of [18F]1 (9 MBq; 0.67 nmol) 3 days after IV injection of tetrazine-derivatised vedolizumab 4 (100 µg; 0.67 nmol)) from 20 to 60 min post tracer injection. All experiments were conducted in SKOV-3 tumor-bearing nude mice.

In conclusion, we have demonstrated for the first time the successful use of a

18

F-labeled small TCO

18

compound, [ F]1, to perform pretargeted immuno-PET imaging. Although some parameters still need to be improved to increase the tumor uptake (e.g., use of a clearing agent, site-specific labeling of the antibody), this pretargeting approach using [18F]1 is a promising starting point to develop antibody-based (radio)pharmaceuticals for instance for diagnosis of brain diseases. Indeed, [18F]1 has the ability to cross the blood-brain barrier, as reported in a previous article. Future studies will now focus on such applications.

Supporting information Material and general methods, syntheses, stability studies in rat plasma, cell binding figure, cell internalization figure, in vivo pretargeted microPET imaging figure, 1H-NMR and

13

C-NMR spectra of

representative compounds.

Abbreviations Cy5-TCO

3-(2-((1E,3E,5E)-5-(1-(6-((3-((((E)-cyclooct-4-en-1-yloxy)carbonyl)amino)propyl)amino)-6-

oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1ium-1-yl)propane-1-sulfonate,

HATU

1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-


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b]pyridinium 3-oxid hexafluorophosphate, HER2 human epidermal growth factor receptor 2, IEDDA inverse-electron-demand Diels–Alder, PEG polyethylene glycol, PET positron emission tomography, TCO trans-cyclooctene.

Acknowledgments The authors thank Julie Cornelis, Pieter Haspeslagh, Ivan Sannen, Jana Hemelaers from the Laboratory for Radiopharmaceutical Research, and Ann Van Santvoort, Tinne Buelens from the MoSAIC facility, for their assistance. This research project received support from IWT Flanders (SBO 130065 MIRIAD).

Author information Corresponding author *Email: [email protected] ORCID Guy Bormans 0000-0002-0335-7190 Emilie Billaud 0000-0001-5783-5082 Notes The authors declare no competing financial interest.

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ToC


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Figure 1. Chemical structures and conceptual schematics of compounds used in this study 170x96mm (300 x 300 DPI)



Scheme 1. Synthesis of tetrazine derivative 8 DCC: N,N′-dicyclohexylcarbodiimide, DIPEA: N,N-diisopropylethylamine, DMAP: 4-(dimethylamino)pyridine, DMF: N,N-dimethylformamide, HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, NHS: N-hydroxysuccinimide, TFA: trifluoroacetic acid.

190x114mm (300 x 300 DPI)


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Figure 2. Cell surface binding and internalization following incubation of HER2-positive SKOV-3 cells with immunoconjugate 3 (1 µM) for 60 min and then with [18F]1 (0.7 MBq.mL-1) for 15 min. Specificity for HER2 was evaluated by using trastuzumab (50 µM) as a blocking agent or by incubating the cells with [18F]1 only (no pretreatment with 3). Selectivity of the IEDDA reaction between trastuzumab-tetrazine derivative 3 and TCO [18F]1 was verified by incubating the cells with non-radioactive Cy5-TCO (50 µM) for 10 min before the addition of [18F]1 . Cells were incubated at 37°C. Data are expressed as mean ± standard error of the mean (N = 3). 288x125mm (300 x 300 DPI)



Figure 3. (A) In vivo pretargeted PET imaging 2 h postinjection (IV) of bioorthogonal [18F]1 (11 MBq; 0.67 nmol), 2 days after IV injection of trastuzumab-tetrazine conjugate 3 (100 µg; 0.67 nmol) in a nude mouse bearing a subcutaneous SKOV-3 xenograft on the right shoulder. Tomographic slices bisect the tumor. The tumor is indicated by an arrow. (B) Time-activity curves from 0 to 60 min after IV injection of [18F]1 (11 MBq; 0.67 nmol), 2 (top, N = 3) or 3 days (bottom, N = 3) after IV injection of trastuzumab-tetrazine conjugate 3 (100 µg; 0.67 nmol) in SKOV-3 tumor-bearing nude mice. Data are expressed as standardized uptake value (SUV). 254x190mm (300 x 300 DPI)


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Figure 4. Tumor-to-muscle activity ratios after IV injection of [18F]1 (11 MBq; 0.67 nmol), 2 or 3 days after IV injection of trastuzumab-tetrazine conjugate 3 (100 µg; 0.67 nmol) are comparable and are significantly higher than in the control experiment (IV injection of [18F]1 (6 MBq) without pretargeting pretreatment) and the non-specific experiment (IV injection of [18F]1 (9 MBq; 0.67 nmol) 3 days after IV injection of tetrazine-derivatised vedolizumab 4 (100 µg; 0.67 nmol)) from 20 to 60 min post tracer injection. All experiments were conducted in SKOV-3 tumor-bearing nude mice. 170x116mm (300 x 300 DPI)


Pretargeted PET Imaging Using a Bioorthogonal 18F-Labeled trans

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