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 v β 3, 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 SiFA lin-modified HBPLs (SiFA = silicon fluoride acceptor), consisting of an MC1R-targeting (GG-Nle-c(DHfRWK)) and an integrin α v β 3-affine peptide (c(RGDfK)), being connected by a symmetrically branching framework including linkers of differing length and composition. Kit-like 18 F-radiolabeling of the HBPLs 1–6 provided the labeled products [18 F]1–[18 F]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 v β 3 (U87MG cells) or the MC1R (B16F10). Based on these results, the most promising compounds [18 F]2, showing the highest affinity to both target receptors (IC 50 (B16F10) = 0.99 ± 0.11 nM, IC 50 (U87MG) = 1300 ± 288 nM), and [18 F]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 [18 F]4, the tracer showed higher absolute tumor uptakes ([18 F]4: 2.58 ± 0.86% ID/g in B16F10 tumors and 3.92 ± 1.31% ID/g in U87MG tumors; [18 F]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 [18 F]2. Thus, [18 F]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 v β 3- and MC1R-bispecific radioligands for the sensitive and specific imaging of malignant melanoma by PET/CT.
With a global incidence increasing over the last decades and being among the tumor types with the most increasing prevalence in Europe, malignant melanoma (MM) is the most aggressive type of skin cancer. The probability of developing the disease is increasing for people with a large number of melanocytic nevi, a fair skin type and genetic predisposition [1,2,3]. Repeated exposure to strong UV (ultraviolet) radiation through recurrent intense sun exposure is the most important environmental risk factor [4]. In most cases, an early diagnosis enables a complete surgical removal and thus the patient to be cured. However, early detection is often not possible since the disease has no particular symptoms, and the tumors can rapidly progress from the fully encapsulated stage to infiltrative growth. In the case of basal membrane penetration, the tumor has access to the blood and lymph vessels, and metastases can be formed in organs or lymph nodes [5]. Since a cure is rarely possible when metastasis has already occurred, an early, very sensitive and specific diagnosis of the disease is of the highest importance. Moreover, the correct staging of the disease is critical, as only, in this case, can an appropriate therapy, having the potential to cure the patient, be chosen.
However, primary diagnosis using positron emission tomography (PET), which has the highest sensitivity compared to other whole-body imaging techniques such as computed tomography (CT) or magnetic resonance imaging (MRI), is often not suitable to correctly identify MM lesions. One drawback of the commonly used radiotracer [18 F]FDG (2-[18 F]fluoro-2-deoxyglucose) is its accumulation in inflamed tissues, giving false-positive results. Furthermore, the detection of slowly growing lesions is often difficult as well, resulting in possible false-negative imaging results [6]. Since the tumor visualization sensitivity and specificity using [18 F]FDG can be low, an early and correct diagnosis and staging are often not possible. An alternative to unspecific, metabolically driven imaging is addressing the tumor by a tumor-specific radiotracer. For this purpose, receptors that are overexpressed in the tumor cell surface are especially useful. In the case of MM, the MC1R is best suited, as this receptor type is overexpressed in about 80% of MM primaries [7,8] and thus is a highly important target structure for MM-specific imaging. However, not all lesions express the MC1R, resulting in an incomplete visualization of the tumor load and thus false staging of the disease. In order to improve the diagnostic imaging of MM and enable an adequate, early and sensitive diagnosis and correct staging, a reliable and sensitive imaging method for MM is needed. Therefore, the development of target-specific accumulating agents that are able to address more than just the MC1R is mandatory.
Such agents should be based on radiolabeled peptides being able to bind with high affinity and specificity to surface receptors overexpressed by malignant cells and thus, enable the distinction between benign and malignant tissue. Ideally, radiolabeled peptides exhibit favorable tumor-to-background ratios, due to their tumor-specific accumulation, and thus produce images of high quality. Furthermore, peptides exhibit low toxicity and immunogenicity, are easily synthetically accessible and can be chemically modified at defined sites. Their pharmacokinetics prove to be very advantageous due to rapid tissue penetration, target accumulation and elimination from non-target tissues [9,10]. Therefore, numerous radiolabeled peptides have been developed for both the diagnosis and therapy of malignancies over the last decades [11,12].
Heterobivalent peptidic ligands (HBPLs), consisting of a radionuclide and two different peptides, each addressing its respective target receptor, have the advantage of a higher target avidity compared to monovalent peptide ligands by being able to bind simultaneously or independently to different target receptors on the tumor surface, resulting in stronger binding to the target cell [13]. Furthermore, HBPLs usually exhibit higher metabolic stability than their respective monomers against peptidases, due to their higher molecular weight and introduction of artificial structural elements [14]. The prerequisite for an HBPL with high tumor visualization potential is at least a moderate binding affinity of each of the included peptides to their target receptors. Ideally, both receptor types should be present in high density to achieve a concomitant binding of both peptide binders of the HBPL; however, the presence of only one target receptor is sufficient to achieve a high tumor uptake [9,15,16,17], resulting in an overall improved imaging sensitivity.
In contrast to HBPLs, monovalent peptides, being able to address only one receptor type and thus only visualize tumors that overexpress this particular receptor, can result in limited tumor visualization sensitivity, as tumor cells can overexpress different receptor types. In such cases of inhomogeneous receptor expression, which can further be caused by tumor dedifferentiation, metastasis or triggered by therapy, the target receptor for the monospecific binder can be absent or present in insufficient density [18,19,20]. This results in an insufficient sensitivity of the peptides’ tumor delineation (Figure 1A). In contrast, HBPLs have the advantage of binding to more than one receptor type and thus exhibit a high tumor visualization efficiency (Figure 1B) [9,20].
Figure 1. Schematic depiction of the concept of HBPL application exemplified by a comparison of a radiolabeled monospecific (A) or heterobivalent peptidic ligand (B) binding to tumor entities overexpressing different receptor types. In the case of (A), no binding is possible since the respective target receptor is only expressed to a low extent. In the case of (B), the HBPL can bind since at least one of the target receptors is expressed on the tumor surface.
For the development of HBPLs, some requirements have to be fulfilled. The peptides have to be modified as little as possible in their chemical structure to preserve their binding affinities to their corresponding receptors. In particular, the pharmacophoric site has to remain unchanged. Furthermore, it is important to determine which receptor types are overexpressed in a tumor entity and thus can be addressed by the radioligand to be developed [9,20]. For this purpose, many studies have been performed within recent years regarding the available receptor types on different human malignancies [21]. The results obtained serve as a guideline for the choice of peptidic receptor ligands, yielding potent tumor-targeting HBPLs with highly sensitive visualization properties.
For MM, the MC1R represents one especially useful target structure for the specific imaging of the disease (vide supra). Another receptor type that is of high potential for MM imaging is integrin α v β 3, as it was shown that this receptor is overexpressed in the blood vessels of many human tumors [22,23,24]. Further studies revealed the involvement of integrin α v β 3 in the progression of the disease and in the change of tumor growth from radial to vertical (thus infiltrative) growth [25,26,27,28,29,30]. Therefore, integrin α v β 3, although overexpressed in all neo-angiogenetic processes, is also an important marker protein for MM targeting.
Thus, HBPLs based on MC1R- and integrin α v β 3-affine peptides would be most promising for visualizing MM during all stages of the disease, enabling a highly sensitive and especially correct assessment of the extent of the disease. This is of crucial importance for choosing the optimal therapy approach, adapted to the extent of the disease: an encapsulated tumor can be treated differently than an infiltratively growing or already metastatic tumor. A high sensitivity to tumor imaging, surely identifying all tumor mass, is thus the prerequisite for the choice of the best-suited therapy option.
So far, the concept to develop an HBPL based on an MC1R-specific peptide ([Cys 3,4,10, DPhe 7, Arg 11]αMSH 3-13) and an integrin α v β 3-affine peptide (c(RGDyD) (cyclic Arg-Gly-Asp-DTyr-Asp)) has only been described once for the radiotracer 99m Tc-RGD-Lys-(Arg 11)CCMSH intended for tumor therapy driven by caspase-3-induced apoptosis induction [31]. The evaluation of this compound was performed in vitro on MC1R-exhibiting B16F1 cells and in vivo in B16F1 melanoma-bearing mice. High binding affinity and tumor uptake, but also a high renal uptake, were detected for this tracer. Therefore, structural modifications are mandatory to obtain an HBPL with more favorable in vivo pharmacokinetics.
In the present study, we developed different radiolabeled MC1R- and α v β 3-bispecific HBPLs. These were based on the α v β 3-affine peptide c(RGDfK) (cyclic Arg-Gly-Asp-DPhe-Lys), showing high stability and integrin target affinity [32], and the macrocyclic lactam GG-Nle-c(DHfRWK) (Gly-Gly-Nle-cyclic Asp-His-DPhe-Arg-Trp-Lys), giving excellent results in terms of MC1R target affinity and stability against proteolytic degradation as well [33,34].
As no HBPLs based on these peptidic ligands have been described so far, we intended to assess the general feasibility of this concept and to develop different HBPLs, consisting of the mentioned peptidic binders, a SiFA lin-moiety (for efficient radiolabeling of the HBPL with the positron-emitting nuclide 18 F) and a varying molecular design. The molecular scaffold for the HBPLs was based on a symmetrical branching unit exhibiting linkers of different lengths and compositions so as to be able to systematically determine the influence of the used linker type and length on the biological parameters of the resulting HBPLs. The developed agents were labeled with 18 F and evaluated in vitro regarding their lipophilicity, stability in human serum and especially their binding affinity to the respective target receptors. Finally, the most promising 18 F-labeled derivatives were evaluated in vivo, in terms of their tumor visualization potential, in an appropriate preclinical tumor model using PET/CT imaging and ex vivo biodistribution experiments.
The molecular design of the target compounds (Figure 2) included two different peptides, each addressing specifically one of the two target receptors—c(RGDfK) for integrin α v β 3 and GG-Nle-c(DHfRWK) for MC1R binding—and was based on the following conditions: (i) The HBPLs should contain a SiFA lin-moiety exhibiting a permanent positive charge. With this SiFA lin building block, the radionuclide 18 F can be efficiently introduced in one step [35]; (ii) the required molecular building blocks should be connected by a small symmetrically branched framework resulting in homogeneous compounds [9,36]; (iii) a lysine spacer should be introduced between the SiFA lin-moiety and the branched framework to achieve a spatial distance between the SiFA lin and the peptides, preventing interference with the peptide–receptor interaction, and to obtain the products in higher radiochemical yields [9,20,37,38]; (iv) as much as possible, the syntheses should be carried out on a solid support to facilitate the assembly of the rather complex target molecules; (v) different linker structures should be used.
