Exploiting the Concept of Multivalency with 68Ga- and 89Zr-Labelled Fusarinine C-Minigastrin Bioconjugates for Targeting CCK2R Expression

Cholecystokinin-2 receptors (CCK2R) are overexpressed in a variety of malignant diseases and therefore have gained certain attention for peptide receptor radionuclide imaging. Among extensive approaches to improve pharmacokinetics and metabolic stability of minigastrin (MG) based radioligands, the concept of multivalency for enhanced tumour targeting has not been investigated extensively. We therefore utilized fusarinine C (FSC) as chelating scaffold for novel mono-, di-, and trimeric bioconjugates for targeting CCK2R expression. FSC-based imaging probes were radiolabelled with positron emitting radionuclides (gallium-68 and zirconium-89) and characterized in vitro (log⁡D, IC50, and cell uptake) and in vivo (metabolic stability in BALB/c mice, biodistribution profile, and microPET/CT imaging in A431-CCK2R/A431-mock tumour xenografted BALB/c nude mice). Improved targeting did not fully correlate with the grade of multimerization. The divalent probe showed higher receptor affinity and increased CCK2R mediated cell uptake while the trimer remained comparable to the monomer. In vivo biodistribution studies 1 h after administration of the 68Ga-labelled radioligands confirmed this trend, but imaging at late time point (24 h) with 89Zr-labelled counterparts showed a clearly enhanced imaging contrast of the trimeric probe compared to the mono- and dimer. Furthermore, in vivo stability studies showed a higher metabolic stability for multimeric probes compared to the monomeric bioconjugate. In summary, we could show that FSC can be utilized as suitable scaffold for novel mono- and multivalent imaging probes for CCK2R-related malignancies with partly improved targeting properties for multivalent conjugates. The increased tumour accumulation of the trimer 24 h postinjection (p.i.) can be explained by slower clearance and increased metabolic stability of multimeric conjugates.


Introduction
Receptor targeting with radiolabelled peptides has become an emerging field in nuclear medicine for early diagnosis and therapy of cancerous diseases [1,2]. The overexpression of cholecystokinin receptor subtype 2 (CCK2R) is involved in various malignancies, such as medullary thyroid carcinoma (MTC), small cell lung cancer (SCLC), and neuroendocrine tumours (NET) [3,4] and therefore represents an interesting target for peptide receptor radionuclide imaging and therapy. In a preclinical study, different derivatives based on human minigastrin (H 2 N-Leu-(Glu) 5 -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 ) for targeting CCK2R expression were investigated in order to find the ideal targeting sequence [5]. This study reported that four C-terminal amino acids (-Trp-Met-Asp-Phe-NH 2 ) are mandatory for selectivity and high affinity towards CCK2R. Different variations are allowed in the N-terminal region to improve in vivo targeting properties and pharmacokinetics, especially kidney uptake which is 2 Contrast Media & Molecular Imaging closely related to the appearance of negatively charged amino acids. Based on this knowledge a variety of MG derivatives have been synthesized by different groups with the aim of reducing kidney but retaining tumour uptake [6]. Further studies have shown that these modifications are unfortunately accompanied by low metabolic stability predominantly for MG11 [7,8]. In vivo stability is a major issue for imaging of receptor expression with small peptide-based molecules. Rapid degradation may lead to decreased tumour uptake and low imaging contrast. Several attempts have been made to overcome this issue, for example, coinjection of the neutral endopeptidase inhibitor phosphoramidon [9] with promising results. Less effort has been spent on a different approach: the design of multivalent constructs. This could increase the probability of receptor target interaction and therefore increase receptor avidity [10,11] as well as promoting the formation of metabolites able to rebind to the receptor. By this, increased apparent stability tumour uptake and therefore improved imaging contrast would be achieved as has been proposed by Carlucci and coworkers [12]. Sosabowski and coworkers were, to the best of our knowledge, the only group so far reporting on a divalent probe (MGD5) for nuclear imaging of CCK2R expression. This tracer consisting of the bifunctional chelator (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA) conjugated to a MG derivative crosslinked via thiol-maleimide to a second sequence of the peptide was radiolabelled with indium-111 for single photon emission tomography (SPECT). It showed increased affinity and tumour uptake compared to its monomeric counterpart 111 In-APH070 [13]. Our group recently reported on fusarinine C (FSC), a cyclic siderophore based bifunctional chelator, providing a scaffold for site-specific conjugation of up to three targeting vectors with excellent complexing properties towards the PET radionuclides gallium-68 and zirconium-89 [14,15]. As positron emission tomography (PET) provides higher resolution compared to SPECT we initiated this study to compare novel mono-, di-, and trimeric FSC-conjugates for targeting CCK2R expression. Due to low metabolic stability minigastrin analogue (MG11) was chosen as model peptide and was conjugated via thiolmaleimide crosslink as shown in Scheme 1. The resulting mono-and multimeric conjugates were radiolabelled with gallium-68 and zirconium-89 followed by in vitro and in vivo characterization.

MALDI-TOF MS.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed on a Bruker microflex6 bench-top MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). Samples were prepared on a microscout target (MSP96 target ground steel BC, Bruker Daltonics) using dried-droplet method and -cyano-4hydroxycinnamic acid (HCCA, Sigma-Aldrich, Handels GmbH, Vienna, Austria) as matrix. All spectra were recorded by summarizing 800 laser shots per spot and Flex Analysis 2.4 software was used for data processing.

Gamma Counter. A 2480 Automatic Gamma Counter
Wizard2 3 (Perkin Elmer, Waltham, MA, USA) was used to measure the radioactivity of samples retrieved from in vitro and in vivo experiments.

Precursor Preparation.
The synthesis of mono-and multivalent FSC-based minigastrin derivatives is presented in detail in the supplementary materials sections.  Figure S1).

In Vitro Characterization
2.9.1. Stability Study. To assess the stability of the radionuclide complex the radiolabelled peptides were diluted with PBS to a concentration of 5 M. Aliquots of 50 L were mixed with PBS (as control), human serum, and a 1.000-fold molar excess over radioligand of either EDTA (pH 7), DTPA (pH 7), or FeCl 3 (pH 5). Then samples were incubated in duplicate at 37 ∘ C up to 4 h for gallium-68 and up to 7 days for zirconium-89 labelled peptides. Samples were analyzed at selected time points via radio-RP-HPLC for 68 Ga-labelled probes and radio-ITLC for 89 Zr-labelled peptides. The ITLC strips were cut into half and measured in the gamma counter to determine the percentage of labelled peptide (origin) to free radionuclide (solvent front).

Distribution Coefficient (log ).
Aliquots (50 L) of 68 Ga-and 89 Zr-labelled mono-, di-, and trimer (10 M) were diluted in 450 L PBS. After adding 500 L octanol the mixture was vortexed with 1400 rpm (MS 3 basic vortexer, IKA, Staufen, Germany) for 15 min at RT followed by centrifugation for 2 min at 4500 rpm. Subsequently, 100 L aliquots of the organic and the aqueous layer were collected and log values were calculated using Excel ( = 3, six replicates) after gamma counter measurement.

Protein Binding.
For protein binding measurement 68 Ga-and 89 Zr-labelled peptides were incubated in PBS as control and fresh human serum and samples were maintained at 37 ∘ C. After 1, 2, and 4 h aliquots (25 L) were analyzed by size exclusion chromatography using MicroSpin G-50 columns (Sephadex G-50, GE Healthcare, Vienna, Austria) according to manufacturer's protocol. The samples were measured in the gamma counter and the percentage between protein-bound (eluate) and free conjugate (column) was calculated.

Precursor Synthesis.
MG11-SH could be obtained in good yield following SPPS protocol.
[Fe]FSC could be extracted from fungal culture in sufficient purity to be used for further modification without additional purification. Acetylation reaction resulted in a mixture of mono-and multiple acetylated derivatives of [Fe]FSC due to three identical primary amines but the desired products were easily accessible via preparative RP-HPLC purification. Functionalization with maleimide linker was straightforward utilizing a NHS-ester strategy and conjugation of up to three targeting vectors was conducted site-specifically via maleimide-thiol crosslink reaction. All intermediates as well as final conjugates were obtained in good yield, with excellent chemical purity (>95%; analytical RP-HPLC, UV absorption at = 220 nm), and corresponding mass analysis was in good agreement with the calculated values.

Radiolabelling.
Mono-and multimeric conjugates were quantitatively labelled with gallium-68 after 5-15 min and after 30-60 min with zirconium-89 at high molar activities and used without further purification. are presented in detail in the Supplementary Materials (Table  S1). Distribution coefficient (log ) values and protein binding data are summarized in Table 1. The results indicate a hydrophilic character of all conjugates but, as expected, clearly showed that the grade of multimerization is accompanied by increased lipophilicity. Binding to serum proteins was low (<10%) for monomeric, moderate (10-25%) for dimeric, and high for (30-50%) trimeric conjugates. Furthermore, protein binding was consistent over time for 68 Ga-labelled mono-and dimer but increased for 68 Ga-trimer whereas all 89 Zr-labelled counterparts showed a slight increase over a period of 4 h.
Competition assays on whole A431-CCK2R ( Figure 1) revealed high binding affinity as the IC 50 values were in the low nanomolar range for all bioconjugates. The affinity of the dimer (0.85 ± 0.15 nM) was approximately 10-fold higher while the monomer (9.7 ± 3.5 nM) and the trimer (8.3 ± 2.2 nM) remained comparable to the reference peptide DOTA-MG11 (9.5 ± 0.5 nM) [17].
Cellular processing of A431-CCK2R cells incubated with 68 Ga-and 89 Zr-labelled mono-and multimeric tracers is summarized in Figure 2. In general, all conjugates showed increasing uptake over time while the unspecific cell bound fraction of corresponding blocking studies remained <1%, thus indicating highly specific receptor-mediated cell uptake. Furthermore the 68 Ga-dimer showed significantly increased uptake after 1 h-incubation compared to the monomer ( = 9.01 × 10 −6 ) and the trimer ( = 0.009) while after 2 hincubation only the uptake of the monomer remained lower 6 Contrast Media & Molecular Imaging

In Vivo Characterization.
Investigations on the in vivo stability are shown in Figure 3. RP-HPLC analysis of the corresponding blood samples showed increasing amount of intact radioligand to be found in following order trimer > dimer > monomer, indicating that multimerization is accompanied with increased metabolic stability. The results of the ex vivo biodistribution studies in double tumour xenografted nude mice are summarized in Table 2 and corresponding tumour-to-organ ratios are presented in Table 3. Monoand dimeric bioconjugates radiolabelled with gallium-68 were rapidly cleared from the bloodstream and showed highly specific tumour targeting properties as the uptake in nontargeted tissue was very low, except kidneys, 1 h after administration of the radiotracers. Furthermore, the dimer 0,0 0,5     showed significantly ( < 0.005) increased tumour uptake compared to the monomer but also higher accumulation in renal tissue. However, the corresponding tumour-to-organ ratios revealed no significant difference in most of the organs. In contrast, the 68 Ga-trimer cleared slowly from the body and showed higher blood level 1 h p.i. This was substantiated as the uptake in malignant tissue was comparable to the monomer 1 h p.i. but increased consistently over time accompanied by very high accumulation renal tissue. In addition, also an elevated accumulation in nontargeted tissue was observed for the 68 Ga-trimer, resulting in significantly lower tumour-toorgan ratios compared to mono-and dimer. In comparison, the respective 89 Zr-labelled counterparts showed a similar behaviour in vivo with somewhat lower tumour uptake 1 h p.i. but consistently higher accumulation in renal tissue. 89 Zr mono-and dimers revealed a trend towards reduced tumourto-organ ratios as compared to the 68 Ga-counterparts. In contrast, over time the 89 Zr-trimer showed a faster elimination from blood and lower unspecific tissue uptake. This resulted in higher tumour-to-organ ratios compared to the 68 Ga-labelled counterpart especially 4 h p.i. and may be attributed to the increased hydrophilic character of this radiotracer. Small animal PET/CT imaging studies confirmed these findings and the results for 68 Ga-labelled conjugates are presented in Figure 4 whereas imaging of the 89 Zrlabelled counterparts is shown in Figure 5. The predominant accumulation of radioactivity found in the kidneys, related to elimination via renal pathway accompanied by tubular reabsorption, was in good agreement with the results of our biodistribution studies and clearly showed that kidney uptake is increased with the grade of multimerization. Furthermore, imaging showed highly specific targeting properties for all conjugates as CCK2 related malignancies were clearly visualized already 1 h after injection without major differences between the bioconjugates. However, imaging at 2 h p.i. for 68 Ga-labelled and that at 24 h p.i for 89 Zr-labelled trivalent probe pronounce the increased uptake in CCK2R expressing tissue.

Discussion
The cyclic siderophore based chelator FSC enables a straightforward multistep synthesis of multivalent imaging probes. Three amines attached to the chelating scaffold of FSC can be utilized for further modifications whereas DOTA, for example, offers only one binding site for conjugation of targeting probes. This results in a different but more symmetrical architecture of FSC-related multivalent bioconjugates. Asymmetric tracer design might not be critical in case of small peptides for targeted imaging but can become an issue in case of multimerization of larger biomolecules (e.g., engineered scaffold proteins, ESP) as the asymmetry can result in less flexibility or increased sterically hindrance at the target interaction site. The suitability for FSC as chelating scaffold for ESP has recently been shown although acetylation of two amines was conducted to design a monovalent affibody construct [18]. Furthermore, FSC can be labelled with gallium-68 and zirconium-89 at RT with sufficient complex stability (Table S1), which is beneficial in case of heat-sensitive molecules. This is also of particular interest for CCK2R targeting peptide derivatives containing methionine as previous studies have shown that heating related oxidation is accompanied by reduced receptor affinity [19]. In contrast, DOTA-conjugates have to be heated for efficient radiolabelling with gallium-68 and in case of oxidation sensitivity of the targeting probe additives (e.g., ascorbic acid) are needed to improve radiochemical purity. Both 68 Ga and 89 Zr FSCconjugates showed high in vivo stability; there was also no significant difference in bone uptake between 68 Ga and 89 Zr counterparts, indicating insignificant in vivo release of 89 Zr, confirming recent findings [15].
Overall FSC-based imaging probes showed high CCK2R binding affinity (Figure 1) with values in the low nanomolar range. The divalent probe showing higher affinity is quite consistent with previous reports on 111 In-MGD5 [11]. Interestingly, increasing the valency from mono-to trimeric constructs, no increase in binding affinity was achieved.
All FSC-based conjugates showed highly specific receptor targeting as demonstrated in vitro ( Figure 2) and also in vivo imaging (Figures 4 and 5) confirmed these findings. Multimerization is accompanied by decreased hydrophilicity and increased binding to serum proteins (Table 2) leading to slower pharmacokinetics in vivo, especially for the trimer. Furthermore, kidney retention was considerably increased by the grade of multimerization (Table 3). This is substantiated by the faster blood clearance of 89 Zr-labelled counterparts due to higher hydrophilicity but increased kidney uptake which may be related to the additional charge introduced as the hexadentate chelator FSC only compensates three of four positive charges of Zr 4+ . Overall, high kidney retention might be critical in case of therapeutic use but is tolerable for diagnostic applications of radiopharmaceuticals [20].
Interestingly, improvement due to multimerization was more pronounced at later time points. While internalization data revealed higher cell uptake at 1 h for the dimer over the trimer, at later time points the trimer showed higher internalization rates. This phenomenon was also seen in vivo where the dimer revealed a higher tumour uptake at 1 h p.i., whereas the uptake increased substantially after 2 and 4 h p.i for the trimer exceeding the values of the dimer at 1 h. p.i.. This in vivo effects may be explained by a slower target accumulation of the trimer but also by the higher protein binding and slow blood clearance that may act as depot to prolong the tracer concentration at the tumour site, improving imaging contrast over time (Table 3). Furthermore, the improved metabolic stability (Figure 3) of multimeric radioligands targeting CCK2R expression, which has been shown in this study for the first time, also may result in the formation of rebinding metabolites enhancing the imaging contrast over time. This hypothesis is substantiated by the slow tumour washout particularly observed for 89 Zr-trimer at 24 h p.i ( Figure 5) making imaging at late timepoints with multivalent peptides tracers radiolabelled with zirconium-89 not per se uninteresting. Additionally, improving metabolic stability by this multivalency-approach might also be highly interesting for non-radiopharmaceutical-based applications such as therapeutic peptides but definitely warrants further investigations regarding formation of metabolites and their target interaction ability.

Conclusion
In this study, novel mono-and multimeric bioconjugates utilizing FSC for radiolabelling with gallium-68 and zirconium-89 for PET applications targeting CCK2R expression were synthesized and evaluated for the first time. The resulting imaging probes showed highly specific receptor targeting characteristics which were only partly improved in terms of binding affinity and in vivo targeting by the grade of multimerization. However, the higher metabolic stability and improved target retention in vivo of multivalent conjugates warrant further investigations on the formation of metabolites with the retained receptor binding ability. Overall this study established FSC as a promising scaffold for the development of mono-and multimeric targeted bioconjugates for molecular imaging with PET.

Disclosure
Dominik Summer's contributions to this study are part of his Ph.D. thesis within the IGDT programme of the Medical University Innsbruck, Austria.

Conflicts of Interest
The authors declare no conflicts of interest.