⁸⁹Zr represents a highly favorable positron emitter for application in immuno-PET (Positron Emission Tomography) imaging. Clinically, the ⁸⁹Zr⁴⁺ ion is introduced into antibodies by complexation with desferrioxamine B. However, producing complexes of limited kinetic inertness. Therefore, several new chelators for ⁸⁹Zr introduction have been developed over the last years. Of these, the direct comparison of the most relevant ones for clinical translation, DFO* and 3,4,3-(LI-1,2-HOPO), is still missing. Thus, we directly compared DFO with DFO* and 3,4,3-(LI-1,2-HOPO) immunoconjugates to identify the most suitable agent stable ⁸⁹Zr-complexation. The chelators were introduced into cetuximab, and an optical analysis method was developed, enabling the efficient quantification of derivatization sites per protein. The cetuximab conjugates were efficiently obtained and radiolabeled with ⁸⁹Zr at 37 °C within 30 min, giving the [⁸⁹Zr]Zr-cetuximab derivatives in high radiochemical yields and purities of >99% as well as specific activities of 50 MBq/mg. The immunoreactive fraction of all ⁸⁹Zr-labeled cetuximab derivatives was determined to be in the range of 86.5–88.1%. In vivo PET imaging and ex vivo biodistribution studies in tumor-bearing animals revealed a comparable and significantly higher kinetic inertness for both [⁸⁹Zr]Zr-3,4,3-(LI-1,2-HOPO)-cetuximab and [⁸⁹Zr]Zr-DFO*-cetuximab, compared to [⁸⁹Zr]Zr-DFO-cetuximab. Of these, [⁸⁹Zr]Zr-DFO*-cetuximab showed a considerably more favorable pharmacokinetic profile with significantly lower liver and spleen retention than [⁸⁹Zr]Zr-3,4,3-(LI-1,2-HOPO)-cetuximab. Since [⁸⁹Zr]Zr-DFO* demonstrates a very high kinetic inertness, paired with a highly favorable pharmacokinetic profile of the resulting antibody conjugate, DFO* currently represents the most suitable chelator candidate for stable ⁸⁹Zr-radiolabeling of antibodies and clinical translation.

Combining two peptides addressing two different receptors to a heterobivalent peptidic ligand (HBPL) is thought to enable an improved tumor-targeting sensitivity and thus tumor visualization, compared to monovalent peptide ligands. In the case of melanoma, the Melanocortin-1 receptor (MC1R), which is stably overexpressed in the majority of primary malignant melanomas, and integrin α_vβ₃, which is involved in lymph node metastasis and therefore has an important role in the transition from local to metastatic disease, are important target receptors. Thus, if a radiolabeled HBPL could be developed that was able to bind to both receptor types, the early diagnosis and correct staging of the disease would be significantly increased. Here, we report on the design, synthesis, radiolabeling and in vitro and in vivo testing of different SiFAlin-modified HBPLs (SiFA = silicon fluoride acceptor), consisting of an MC1R-targeting (GG-Nle-c(DHfRWK)) and an integrin α_vβ₃-affine peptide (c(RGDfK)), being connected by a symmetrically branching framework including linkers of differing length and composition. Kit-like ¹⁸F-radiolabeling of the HBPLs 1–6 provided the labeled products [¹⁸F]1–[¹⁸F]6 in radiochemical yields of 27–50%, radiochemical purities of ≥95% and non-optimized molar activities of 17–51 GBq/μmol within short preparation times of 25 min. Besides the evaluation of radiotracers regarding log_D(7.4) and stability in human serum, the receptor affinities of the HBPLs were investigated in vitro on cell lines overexpressing integrin α_vβ₃ (U87MG cells) or the MC1R (B16F10). Based on these results, the most promising compounds [¹⁸F]2, showing the highest affinity to both target receptors (IC_{50 (B16F10)} = 0.99 ± 0.11 nM, IC_{50 (U87MG)} = 1300 ± 288 nM), and [¹⁸F]4, exhibiting the highest hydrophilicity (log_D(7.4) = −1.39 ± 0.03), were further investigated in vivo and ex vivo in a xenograft mouse model bearing both tumors. For both HBPLs, clear visualization of B16F10, as well as U87MG tumors, was feasible. Blocking studies using the respective monospecific peptides demonstrated both peptide binders of the HBPLs contributing to tumor uptake. Despite the somewhat lower target receptor affinities (IC_{50 (B16F10)} = 6.00 ± 0.47 nM and IC_{50 (U87MG)} = 2034 ± 323 nM) of [¹⁸F]4, the tracer showed higher absolute tumor uptakes ([¹⁸F]4: 2.58 ± 0.86% ID/g in B16F10 tumors and 3.92 ± 1.31% ID/g in U87MG tumors; [¹⁸F]2: 2.32 ± 0.49% ID/g in B16F10 tumors and 2.33 ± 0.46% ID/g in U87MG tumors) as well as higher tumor-to-background ratios than [¹⁸F]2. Thus, [¹⁸F]4 demonstrates to be a highly potent radiotracer for the sensitive and bispecific imaging of malignant melanoma by PET/CT imaging and impressively illustrates the suitability of the underlying concept to develop heterobivalent integrin α_vβ₃- and MC1R-bispecific radioligands for the sensitive and specific imaging of malignant melanoma by PET/CT.

Recently, anionic charges were found to negatively influence the in vitro gastrin-releasing peptide receptor (GRPR) binding parameters of dually radioisotope and fluorescent dye labeled GRPR-specific peptide dimers. From this, the question arose if this adverse impact on in vitro GRP receptor affinities could be mitigated by a higher valency of peptide multimerization. For this purpose, we designed two different hybrid multimodal imaging units (MIUs), comprising either one or two click chemistry-compatible functional groups and reacted them with PESIN (PEG₃-BBN_7–14, PEG = polyethylene glycol) dimers to obtain a dually labeled peptide homodimer or homotetramer. Using this approach, other dually labeled peptide monomers, dimers, and tetramers can also be obtained, and the chelator and fluorescent dye can be adapted to specific requirements. The MIUs, as well as their peptidic conjugates, were evaluated in terms of their photophysical properties, radiolabeling efficiency with ⁶⁸Ga and ⁶⁴Cu, hydrophilicity, and achievable GRP receptor affinities. Here, the hydrophilicity and the GRP receptor binding affinities were found to be especially strongly influenced by the number of negative charges and peptide copies, showing log_D (1-octanol-water-distribution coefficient) and IC₅₀ (half maximal inhibitory concentration) values of −2.2 ± 0.1 and 59.1 ± 1.5 nM for the homodimer, and −1.9 ± 0.1 and 99.8 ± 3.2 nM for the homotetramer, respectively. From the obtained data, it can be concluded that the adverse influence of negatively charged building blocks on the in vitro GRP receptor binding properties of dually labeled PESIN multimers can, at least partly, be compensated for by the number of introduced peptide binding motives and the used molecular design.

In the context of hybrid multimodal imaging agents for gastrin releasing peptide receptor (GRPR) targeting, a correlation between the net charge and the receptor affinity of the agents was recently found. In particular, a decrease in in vitro GRPR binding affinity was observed in case of an increasing number of negative charges for dually labeled GRPR-specific peptide dimers suited for positron emission tomography and optical imaging (PET/OI). This adverse influence of anionic charges could be in part compensated by a higher valency of peptide multimerization. However, it remains unknown whether this adverse effect of anionic charges is limited to peptide multimers or if it is also found or even more pronounced when GRPR-specific peptide monomers are dually labeled with fluorescent dye and chelating agent/radionuclide. Moreover, it would be important to know if this effect is limited to GRPR-specific agents only or if these observations also apply to other dually labeled peptides binding to other receptor types. To address these questions, we synthesized hybrid labels, comprising a chelator, different fluorescent dyes carrying different net charges and a functional group for bioconjugation and introduced them into different peptides, specifically targeting the GRPR, the melanocortin-1 receptor (MC1R) and integrin α_vβ₃. The synthesized conjugates were evaluated with regard to their chemical, radiochemical, photophysical and receptor affinity properties. It was found that neither the ⁶⁸Ga-radiolabeling nor the fluorescence characteristics of the dyes were altered by the conjugation of the MIUs to the peptides. Further, it was confirmed that the net number of anionic charges has a negative effect on the GRPR-binding affinity of the GRPR-targeting MIU-peptide monomer conjugates and that this same effect was also found to the same extent for the other receptor systems studied.