Hybrid Imaging Agents for Pretargeting Applications Based on Fusarinine C—Proof of Concept

Hybrid imaging combining the beneficial properties of radioactivity and optical imaging within one imaging probe has gained increasing interest in radiopharmaceutical research. In this study, we modified the macrocyclic gallium-68 chelator fusarinine C (FSC) by conjugating a fluorescent moiety and tetrazine (Tz) moieties. The resulting hybrid imaging agents were used for pretargeting applications utilizing click reactions with a trans-cyclooctene (TCO) tagged targeting vector for a proof of principle both in vitro and in vivo. Starting from FSC, the fluorophores Sulfocyanine-5, Sulfocyanine-7, or IRDye800CW were conjugated, followed by introduction of one or two Tz motifs, resulting in mono and dimeric Tz conjugates. Evaluation included fluorescence microscopy, binding studies, logD, protein binding, in vivo biodistribution, µPET (micro-positron emission tomography), and optical imaging (OI) studies. 68Ga-labeled conjugates showed suitable hydrophilicity, high stability, and specific targeting properties towards Rituximab-TCO pre-treated CD20 expressing Raji cells. Biodistribution studies showed fast clearance and low accumulation in non-targeted organs for both SulfoCy5- and IRDye800CW-conjugates. In an alendronate-TCO based bone targeting model the dimeric IRDye800CW-conjugate resulted in specific targeting using PET and OI, superior to the monomer. This proof of concept study showed that the preparation of FSC-Tz hybrid imaging agents for pretargeting applications is feasible, making such compounds suitable for hybrid imaging applications.


Introduction
Various imaging modalities have evolved as valuable tools for molecular imaging of human diseases. When used as a stand-alone technique, every method shows benefits and limitations [1] so combining different modalities, namely dual-modality-(DMI) or hybrid imaging (HI), is a field with increasing attraction. Optical imaging (OI), for example, is characterized by excellent sensitivity to acquire morphological information but poor tissue penetration limits its applicability for non-invasive

Synthesis
The FSC-based Tz-bearing hybrid imaging agents were accessible by a straightforward threefour-step synthesis starting from the macrocyclic chelator [Fe]FSC in a similar approach as described in [18,19]. The monomeric conjugates were prepared by initial coupling of the Tetrazine-PEG5-NHS ester to the mono acetyl protected form of FSC, [Fe]MAFC, the dimeric conjugates by starting from [Fe]FSC. Using equal molar amounts the predominant products were [Fe]FSC-(Tetrazine-PEG5)2 or [Fe]MAFC-Tetrazine-PEG5, which were isolated by high-performance liquid chromatography (HPLC), whereby for [Fe]FSC yields were lower due to formation of [Fe]FSC-(Tetrazine-PEG5). The resulting conjugates were straight forward coupled with the respective Dyes by O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorphosphate (HATU) activation and finally iron was removed. The conjugates could be obtained in acceptable yield i.e., 30-60% for monomeric and 40-60% for dimeric FSC-based Tz hybrid imaging agents in sufficient purity (>90%) determined by analytical reversed phase (RP)-HPLC using UV absorption at λ = 220 nm.

Radiolabeling
Radiolabeling with the 68 Ge/ 68 Ga-generator derived radiometal gallium-68 was conducted at ambient temperature within five minutes and radiochemical yields greater than 98% determined by radio-RP-HPLC and radio-ITLC (instant thin layer chromatography) could be achieved-as exemplarily shown in Figure 1.

Synthesis
The FSC-based Tz-bearing hybrid imaging agents were accessible by a straightforward three-four-step synthesis starting from the macrocyclic chelator [Fe]FSC in a similar approach as described in [18,19]. The monomeric conjugates were prepared by initial coupling of the Tetrazine-PEG 5 -NHS ester to the mono acetyl protected form of FSC, [Fe]MAFC, the dimeric conjugates by starting from [Fe]FSC. Using equal molar amounts the predominant products were [Fe]FSC-(Tetrazine-PEG 5 ) 2 or [Fe]MAFC-Tetrazine-PEG 5 , which were isolated by high-performance liquid chromatography (HPLC), whereby for [Fe]FSC yields were lower due to formation of [Fe]FSC-(Tetrazine-PEG 5 ). The resulting conjugates were straight forward coupled with the respective Dyes by O-(7-Azabenzotriazol-1-yl)-N,N,N ,N -tetramethyluronium-hexafluorphosphate (HATU) activation and finally iron was removed. The conjugates could be obtained in acceptable yield i.e., 30-60% for monomeric and 40-60% for dimeric FSC-based Tz hybrid imaging agents in sufficient purity (>90%) determined by analytical reversed phase (RP)-HPLC using UV absorption at λ = 220 nm.

Radiolabeling
Radiolabeling with the 68 Ge/ 68 Ga-generator derived radiometal gallium-68 was conducted at ambient temperature within five minutes and radiochemical yields greater than 98% determined by radio-RP-HPLC and radio-ITLC (instant thin layer chromatography) could be achieved-as exemplarily shown in Figure 1.

In Vitro Characterization
The results of the distribution coefficient logD and protein binding studies are presented in Table 1. All 68 Ga-labeled conjugates showed reasonable hydrophilicity with values ranging from -1.92 for the most lipophilic (SulfoCy7-MAFC-PEG 5 -Tz) to -2.85 for the most hydrophilic conjugate (SulfoCy5-MAFC-PEG 5 -Tz). Interestingly, by replacing the acetyl residue by a second PEG 5 -Tz moiety the hydrophilicity was altered for the SulfoCy5-conjugate while for the corresponding IRDye800CW-conjugate it remained the same. The protein binding of the 68 Ga-labeled FSC-based hybrid imaging agents was consistent over time and ranged from intermediate (30-50%) to high (50-70%). Introducing a SulfoCy5-or -Cy7 residue to the MAFC-Tz scaffold showed a comparable protein binding while the conjugation of the IRDye800CW led to significantly increased values. Interestingly, the replacement of the acetyl moiety by a PEG 5 -Tz residue did not increase the protein binding compared to the monomeric Tz conjugates. However, this is consistent with the results of a previous study where increasing the number of PEG 5 -Tz by chelator scaffolding did not increase the protein binding [19].

In Vitro Characterization
The results of the distribution coefficient logD and protein binding studies are presented in Table  1. All 68 Ga-labeled conjugates showed reasonable hydrophilicity with values ranging from -1.92 for the most lipophilic (SulfoCy7-MAFC-PEG5-Tz) to -2.85 for the most hydrophilic conjugate (SulfoCy5-MAFC-PEG5-Tz). Interestingly, by replacing the acetyl residue by a second PEG5-Tz moiety the hydrophilicity was altered for the SulfoCy5-conjugate while for the corresponding IRDye800CWconjugate it remained the same. The protein binding of the 68 Ga-labeled FSC-based hybrid imaging agents was consistent over time and ranged from intermediate (30-50%) to high (50-70%). Introducing a SulfoCy5-or -Cy7 residue to the MAFC-Tz scaffold showed a comparable protein binding while the conjugation of the IRDye800CW led to significantly increased values. Interestingly, the replacement of the acetyl moiety by a PEG5-Tz residue did not increase the protein binding compared to the monomeric Tz conjugates. However, this is consistent with the results of a previous study where increasing the number of PEG5-Tz by chelator scaffolding did not increase the protein binding [19].   The results of cell binding studies of the 68 Ga-labeled FSC-based hybrid imaging agents on CD20-expressing Raji cells pre-treated with Rituximab(RTX)-TCO-modified or non-modified RTX are summarized in Figure 2. All conjugates showed highly specific targeting properties with ratios of specifically to non-specifically bound radioligand ranging from 3 to 5. The binding of the 68 Ga-labeled  [19]. The binding of the dimeric DMI agents radiolabeled with gallium-68 was significantly higher (p < 0.005) in comparison to their monomeric counterparts and resulted to be 5.91 ± 1.62% for [ 68 Ga]Ga-SulfoCy5-FSC-(PEG 5 -Tz) 2 and 4.59 ± 1.45% for [ 68 Ga]Ga-IRDye800CW-FSC-(PEG 5 -Tz) 2 but was significantly lower (p < 0.0005) compared to the 68 Ga-labeled non-fluorescent conjugate [ 68 Ga]Ga-MAFC-(PEG 5 -Tz) 2 (7.35 ± 0.50%) [19]. Furthermore, the unspecific binding was significantly increased (p < 0.005) also when comparing 68 Ga-labeled mono-and dimeric FSC-based DMI agents.

Biodistribution Studies
The ex vivo biodistribution profile of the 68 Ga-labeled FSC-based hybrid imaging agents in healthy BALB/C mice 1 h post injection (p.i.) is summarized in Figure 4. Except from the SulfoCy7-conjugate all imaging probes showed relatively fast blood clearance and low retention in muscle and bone indicating suitable properties for imaging applications matching the short half-life of Gallium-68. The SulfoCy7-conjugate also revealed higher activities in all organs compared to their SulfoCy5 and IRDye800CW counterparts, matching the highest lipophilicity of this conjugate, which was therefore not further explored in imaging studies. Comparable tissue activity was found comparing Sulfo-Cy5 and IRdye800CW conjugates for most organs, except for kidneys where values for IRDye800CW were considerably higher (>20%IA/g). Monomeric MAFC-PEG 5 -Tz conjugates showed comparable biodistribution to non-dye conjugated FSC-based tetrazines [19] and considerably lower activities especially in liver and spleen (~2%IA/g) as compared to FSC-(PEG 5 -Tz) 2 conjugates with around 10%IA/g. Lung retention was relatively high for all compounds (~5-10%IA/g), no specific reason for this phenomenon could be found. Molecules 2020, 25, x 6 of 15

Biodistribution Studies
The ex vivo biodistribution profile of the 68 Ga-labeled FSC-based hybrid imaging agents in healthy BALB/C mice 1 h post injection (p.i.) is summarized in Figure 4. Except from the SulfoCy7conjugate all imaging probes showed relatively fast blood clearance and low retention in muscle and bone indicating suitable properties for imaging applications matching the short half-life of Gallium-68. The SulfoCy7-conjugate also revealed higher activities in all organs compared to their SulfoCy5 and IRDye800CW counterparts, matching the highest lipophilicity of this conjugate, which was therefore not further explored in imaging studies. Comparable tissue activity was found comparing Sulfo-Cy5 and IRdye800CW conjugates for most organs, except for kidneys where values for IRDye800CW were considerably higher (>20%IA/g). Monomeric MAFC-PEG5-Tz conjugates showed comparable biodistribution to non-dye conjugated FSC-based tetrazines [19] and considerably lower activities especially in liver and spleen (~2%IA/g) as compared to FSC-(PEG5-Tz)2 conjugates with around 10%IA/g. Lung retention was relatively high for all compounds (~5-10%IA/g), no specific reason for this phenomenon could be found.

Imaging Studies
A simple pretargeting model using bone targeting TCO-alendronate was chosen for a straight forward proof of concept in vivo. PET/CT images revealed specific uptake of both IRDye800CW conjugates in bone structures of TCO-alendronate pre-treated mice, whereas no uptake was seen in mice treated with alendronate alone, proving the in vivo specificity of both constructs. Both conjugates revealed predominant renal excretion and good contrast 1 h p.i. Figure 5 shows images of [ 68 Ga]Ga-IRDdye800CW-FSC-(PEG5-Tz)2 revealing high accumulation in joints and spine with excellent contrast and no uptake in the alendronate control images. This specific accumulation could be

Imaging Studies
A simple pretargeting model using bone targeting TCO-alendronate was chosen for a straight forward proof of concept in vivo. PET/CT images revealed specific uptake of both IRDye800CW conjugates in bone structures of TCO-alendronate pre-treated mice, whereas no uptake was seen in mice treated with alendronate alone, proving the in vivo specificity of both constructs. Both conjugates revealed predominant renal excretion and good contrast 1 h p.i. Figure 5 shows images of [ 68 Ga]Ga-IRDdye800CW-FSC-(PEG 5 -Tz) 2 revealing high accumulation in joints and spine with excellent contrast and no uptake in the alendronate control images. This specific accumulation could be confirmed by optical imaging of excised bones with a high accumulation in the joint area, which is not seen in the alendronate control animal. Comparable images of [ 68 Ga]Ga-IRDye800CW-MAFC-PEG 5 -Tz are shown in supplementary data ( Figure S1) indicating less pronounced uptake in comparison with its monomeric counterpart both in PET/CT and optical images, however without being statistically significant. This is in line with the higher in vitro binding ( Figure 2) and our findings when comparing non derivatized mono-and dimeric Tetrazine-FSC conjugates [19], showing that dimerization has a positive effect on in vivo targeting properties. Overall, this proves that dye-conjugated tetrazines based on the FSC scaffold can be used for pretargeting applications combining PET and optical imaging. Further studies, ideally in appropriate pretargeting tumor models, are required to judge the full potential of these compounds for tumor imaging and image-guided procedures. This is to our knowledge the first report on combining PET and NIRF imaging based on a single tetrazine based pretargeting vector. The FSC scaffold, which is a versatile chelator for Gallium-68, was well suited to develop this approach. In contrast to other attempts, where the dye was conjugated to an antibody [16,17], our approach allows a DMI application without modifying the actual targeting vector, thereby avoiding separate development and characterization of the two versions with and without the dye conjugate.

Analytics
Analytical [radio]-RP-HPLC. Reversed-phase high-performance liquid chromatography analysis was performed with the following instrumentation: UltiMate 3000 RS UHPLC pump, This is to our knowledge the first report on combining PET and NIRF imaging based on a single tetrazine based pretargeting vector. The FSC scaffold, which is a versatile chelator for Gallium-68, was well suited to develop this approach. In contrast to other attempts, where the dye was conjugated to an antibody [16,17], our approach allows a DMI application without modifying the actual targeting vector, thereby avoiding separate development and characterization of the two versions with and without the dye conjugate.

Synthesis
General. All chemicals and solvents were obtained as reagent grade from commercial sources unless otherwise stated. Trans-Cyclooctene-NHS ester and Tetrazine-PEG 5 -NHS ester were purchased from Click Chemistry Tools (Scottsdale, AZ, USA). Rituximab (MabThera ® , Roche Pharma AG, Grenzach-Wyhlen, Germany) was a kind gift from the University Hospital of Innsbruck. The fluorescent dyes, SulfoCyanine5-NHS ester and SulfoCyanine7 carboxylic acid were obtained from Lumiprobe GmbH (Hannover, Germany) while IRDye800CW carboxylic acid was purchased from LI-COR Biosciences GmbH (Bad Homburg, Germany).

[Fe]fusarinine C ([Fe]FSC) and [Fe]N-Monoacetylfusarinine C ([Fe]MAFC)
The macrocyclic iron protected precursors were obtained according to previously published procedures [19]. Briefly, [Fe]FSC was directly isolated from fungal culture in high yield and sufficient chemical purity (>90%, UV-Vis, λ = 220 nm  4 µmol) was dissolved in 500 µL dry DMF and after pH adjustment with DIPEA (pH 9) the mixture was stirred for 30 min at RT. Tetrazine-PEG 5 -NHS (1 equivalent) was dissolved in 200 µL anhydrous DMF and was slowly added dropwise to the solution over a period of 15 min. After 2 h reaction time at ambient temperature the organic solvent was concentrated in vacuo and purified by preparative RP-HPLC using gradient B to give [Fe]FSC-(PEG 5 -Tz) 2 (t R = 32.9 min) and [Fe]MAFC-PEG 5 -Tz (t R = 26.9 min) as red-brown colored solid after lyophilization. Analytical data: The remaining half of the reaction mixture (500 µL) was used for demetallation. Therefore, 1 mL of disodium EDTA (Na 2 EDTA, 200 mM) was added and the mixture was stirred for 4 h at RT to completely remove the iron from the conjugates followed by preparative RP-HPLC purification to give intensively green to blue colored solids after freeze drying. Analytical data: •

Synthesis of Dimeric FSC-based Tz Hybrid Imaging Agents
Conjugation of the fluorescent dye to the dimeric FSC-based Tz-ligand was performed as described above using 1.0 mg of [Fe]FSC-(PEG 5 -Tz) 2 (0.57 µmol) as starting material. After successful conjugation, demetallation was performed as described above followed by purification via preparative RP-HPLC. Analytical data: Anti-CD20 monoclonal antibody Rituximab was modified as previously reported [19]. Briefly, the antibody solution (Mabthera ® , 10 mg/mL, Roche Pharma AG, Grenzach-Wyhlen, Germany) was allowed to react at 4 • C overnight in 0.1 M NaHCO 3 solution with 20 molar equivalent of TCO-NHS ester dissolved in DMSO followed by size exclusion column purification via PD-10 (GE Healthcare Vienna, Austria) to give RTX-TCO in PBS (2.5 mg/mL).

Radio-ITLC
Instant thin layer chromatography (ITLC) analysis was performed using TLC-SG strips (Varian, Lake Forest, CA, USA) as stationary phase and 0.1 M sodium citrate solution (pH 5) or 1 M ammonium acetate/methanol (1:1, v/v) with a pH of 6.8 as mobile phase. The strips were analyzed using a TLC scanner (Scan-RAM ™ , LabLogic, Sheffield, UK).

Protein Binding
The ability of the 68 Ga-labeled conjugates to bind to serum proteins was determined by size exclusion chromatography. Aliquots (50 µL, n = 3) of the radioligand solution (~10 µM) were incubated in 450 µL freshly prepared human serum as well as 450 µL PBS as control and the mixtures were maintained at 37 • C. After 1, 2, and 4 h, aliquots (25 µL) were transferred to illustra MicroSpin G-50 columns (GE Healthcare Vienna, Austria) and the percentage of protein-bound (eluate) and non-bound (column) conjugate was calculated.

Cell-Binding Studies
CD20-expressing Raji cells (humanoid lymphoblast-like B-lymphocyte cells) were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). The cells were cultured in tissue culture flasks (Cellstar; Greiner Bio-One, Kremsmuenster, Austria) using RPMI-1640 medium with 10% (v/v) fetal bovine serum (FBS) as supplement (Invitrogen Corporation, Lofer, Austria). For cell binding studies 10 × 10 6 cells were washed twice with fresh medium, diluted with PBS to a final concentration of 1 × 10 6 cells per mL and 500 µL of cell suspension was transferred to Eppendorf tubes. Hereafter, 50 µL of RTX-TCO or non-modified RTX as negative control (both 0.5 µM) was added and the cell suspension was maintained at 37 • C under gentle shaking. After 1 h the suspension was centrifuged (2 min, 11 × 10 3 rcf), the supernatant was discarded and the cells were washed twice with 600 µL PBS and finally resuspended with 450 µL PBS. Subsequently 50 µL of the radioligand solution (22 nM) was added and the suspension was incubated for 30 min at 37 • C. After centrifugation and two washing steps with 600 µL PBS, the cells were resuspended in 500 µL PBS and transferred to polypropylene vials for gamma counter measurement followed by calculation of cell-associated activity in comparison to the total activity applied (n = 3, six replicates).

Fluorescence Microscopy
CD20-expressing Raji cells were prepared and pre-treated with modified or non-modified RTX as described above (cell-binding studies). Instead of the radioligand, 50 µL (22 nM) of iron protected SulfoCyanine5-[Fe]MAFC-PEG 5 -Tz was added and the cells were treated according to the cell-binding studies with the 68 Ga-labeled counterparts. After resuspension fluorescence microscopy was performed using a laser (λ = 561 nm) for excitation of the fluorescent dye. The imaging was carried out with a spinning-disc confocal microscopic system (Ultra VIEW VoX, PerkinElmer, Waltham, MA, USA) linked to a Zeiss AxioObserver Z1 inverted microscope (Zeiss, Oberkochen, Germany). Images were acquired with Volocity software (PerkinElmer) utilizing a 63× oil immersion objective (numerical aperture 1.42). The images show z-stacks (n = 4; 1 µm spacing). Cell morphology was visualized by adding fluorescently labeled wheat germ agglutinin (Alexa Fluor ® 488 WGA, ThermoFischer Scientific, Vienna, Austria).

Biodistribution Studies
Biodistribution studies of the FSC-based hybrid imaging agents radiolabeled with Gallium-68 were performed using healthy five-week-old female BALB/c mice (Charles River Laboratories, Sulzfeld, Germany). Animals (n = 3) were injected via lateral tail vein with 1 nmol of conjugate and a total activity of approximately 6 MBq. Mice were sacrificed by cervical dislocation 1 h p.i. followed by collection of the main organs and tissue, subsequent gamma counter measurement and calculation of the percentage of injected activity per gram tissue (% IA/g).