Ingrid Dijkgraaf

Comparison of a monomeric and dimeric radiolabeled RGD-peptide for tumor targeting.

The alpha v beta 3 integrin, a transmembrane heterodimeric protein expressed on sprouting endothelial cells, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin. Growing malignant tumors continuously require angiogenesis. As a result, alpha v beta 3 is preferentially expressed in growing tumors and is a potential target for radiolabeled RGD-peptides. In this study we compared the tumor targeting characteristics of a monomeric radiolabeled RGD-peptide with those of a dimeric analogue. Both peptides were radiolabeled with 99mTc via the hydrazinoni-cotinamid (= HYNIC) moiety to form 99mTc-HYNIC-c(RGDfK) and 99mTc-HYNIC-E-[c(RGDfK)]2. In vitro, the IC50 showed a 10-fold higher affinity of the dimer for the alpha v beta 3 integrin as compared to the monomer (0.1 vs. 1.0 nM). In athymic female BALB/c mice with subcutaneously growing OVCAR-3 ovarian carcinoma xenografts, tumor uptake peaked at 5.8 +/- 0.7% ID/g and 5.2 +/- 0.6% ID/g for the dimer and the monomer, respectively. At 1, 2, and 4 h postinjection (p.i.) uptake of the dimer in the tumor was significantly higher than that of the monomeric analogue. Tumor-to-blood ratios were highest at 24 h p.i. at a value of 63 for both compounds. At all timepoints kidney retention of the dimer was significantly higher as compared to kidney retention of the monomer. In conclusion, in this mouse model the dimeric RGD-peptide showed better retention in the tumor than the monomeric analogue, most likely due to the bivalent interaction with the target cell. Furthermore, kidney retention of the dimeric peptide was higher than that of the monomeric peptide.

PET of CXCR4 expression by a (68)Ga-labeled highly specific targeted contrast agent.

The overexpression of the chemokine receptor CXCR4 plays an important role in oncology, since together with its endogenous ligand, the stromal cell–derived factor (SDF1-α), CXCR4 is involved in tumor development, growth, and organ-specific metastasis. As part of our ongoing efforts to develop highly specific CXCR4-targeted imaging probes and with the aim to assess the suitability of this ligand for first proof-of-concept studies in humans, we further evaluated the new 68Ga-labeled high-affinity cyclic CXCR4 ligand, 68Ga-CPCR4-2 (cyclo(D-Tyr1-[NMe]-D-Orn2-[4-(aminomethyl) benzoic acid,68Ga-DOTA]-Arg3-2-Nal4-Gly5)). Methods: Additional biodistribution and competitions studies in vivo, dynamic PET studies, and investigations on the metabolic stability and plasma protein binding were performed in nude mice bearing metastasizing OH1 human small cell lung cancer xenografts. CXCR4 expression on OH1 tumor sections was determined by immunohistochemical staining. Results: natGa-CPCR4-2 exhibits high CXCR4 affinity with a half maximum inhibitory concentration of 4.99 ± 0.72 nM. 68Ga-CPCR4-2 showed high in vivo stability and high and specific tumor accumulation, which was reduced by approximately 80% in competition studies with AMD3100. High CXCR4 expression in tumors was confirmed by immunohistochemical staining. 68Ga-CPCR4-2 showed low uptake in nontumor tissue and particularly low kidney accumulation despite predominant renal excretion, leading to high-contrast delineation of tumors in small-animal PET studies. Conclusion: The small and optimized cyclic peptide CPCR4-2 labeled with 68Ga is a suitable tracer for targeting and imaging of human CXCR4 receptor expression in vivo. The high affinity for CXCR4, its in vivo stability, and the excellent pharmacokinetics recommend the further evaluation of 68Ga-CPCR4-2 in a proof-of-concept study in humans.

Design, Synthesis, and Functionalization of Dimeric Peptides Targeting Chemokine Receptor CXCR4

The chemokine receptor CXCR4 is a critical regulator of inflammation and immune surveillance, and it is specifically implicated in cancer metastasis and HIV-1 infection. On the basis of the observation that several of the known antagonists remarkably share a C(2) symmetry element, we constructed symmetric dimers with excellent antagonistic activity using a derivative of a cyclic pentapeptide as monomer. To optimize the binding affinity, we investigated the influence of the distance between the monomers and the pharmacophoric sites in the synthesized constructs. The affinity studies in combination with docking computations support a two-site binding model. In a final step, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was introduced as chelator for (radio-)metals, thus allowing to exploit these compounds as a new group of CXCR4-binding peptidic probes for molecular imaging and endoradiotherapeutic purposes. Both the DOTA conjugates and some of their corresponding metal complexes retain good CXCR4 affinity, and one (68)Ga labeled compound was studied as PET tracer.

PET of Tumors Expressing Gastrin-Releasing Peptide Receptor with an 18F-Labeled Bombesin Analog

The gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer. Bombesin (BBN) is a neurotransmitter of 14 amino acids and binds with selectivity and with high affinity to GRPRs. We have synthesized a NOTA-conjugated bombesin derivative, NOTA-8-Aoc-BBN(7-14)NH2, to label this analog with 18F using the new Al18F method. In this study, the GRPR-targeting potential of 18F-labeled NOTA-8-Aoc-BBN(7-14)NH2 was studied using 68Ga-NOTA-8-Aoc-BBN(7-14)NH2 as a reference. Methods: The NOTA-conjugated bombesin analog was synthesized and radiolabeled with 68Ga or 18F. For 18F labeling, we used our new 1-pot, 1-step method. The labeled product was purified by reversed-phase high-performance liquid chromatography. The log P values of the radiotracers were determined. The tumor-targeting characteristics of the compounds were assessed in mice with subcutaneously growing PC-3 xenografts. GRPR-binding specificity was studied by coinjection of an excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH2. Small-animal PET/CT images were acquired. Results: NOTA-8-Aoc-BBN(7-14)NH2 could be efficiently labeled with 18F or with 68Ga. NOTA-8-Aoc-BBN(7-14)NH2 was labeled with 18F in a single step, with 50%–90% yield. Radiolabeling, including purification, was performed in 45 min and resulted in a specific activity of greater than 10 GBq/μmol. The log P values of 18F- and 68Ga-labeled NOTA-8-Aoc-BBN(7-14)NH2 were −1.47 ± 0.05 and −1.98 ± 0.03, respectively. In mice, both radiolabeled compounds cleared rapidly from the blood (<0.07 percentage injected dose per gram at 1 h after injection), mainly via the kidneys. At 1 h after injection, the uptake of 18F- and 68Ga-labeled NOTA-8-Aoc-BBN(7-14)NH2 in the PC-3 tumors was 2.15 ± 0.55 and 1.24 ± 0.26 percentage injected dose per gram, respectively. GRPR-binding specificity was demonstrated by reduced tumor uptake of radiolabeled NOTA-8-Aoc-BBN(7-14)NH2 after coinjection of a 100-fold excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH2 peptide. The accumulation of 18F-NOTA-8-Aoc-BBN(7-14)NH2 in the subcutaneous PC-3 tumors could be visualized via small-animal PET. Conclusion: NOTA-8-Aoc-BBN(7-14)NH2 could be labeled rapidly and efficiently with 18F using a 1-pot, 1-step method. Radiolabeled NOTA-8-Aoc-BBN(7-14)NH2 specifically accumulated in the GRPR-expressing PC-3 tumors and should be evaluated clinically.

Introduction of functional groups into peptides via N-alkylation.

An optimized protocol for the mild and selective Fukuyama-Mitsunobu reaction was used for mono- and di- N-alkylation on solid support. Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary amines of a linear peptide. N-Alkylation can also be used to implement alkyl chains carrying (protected) functionalities suited for subsequent modification. Applicability of this method is demonstrated by various N-alkylated analogues of a cyclic CXCR4 receptor antagonist originally developed by Fujii et. al.

Radionuclide imaging of tumor angiogenesis.

Angiogenesis is a multistep process regulated by pro- and antiangiogenic factors. In order to grow and metastasize, tumors need a constant supply of oxygen and nutrients. For growth beyond 1-2 mm in size, tumors are dependent on angiogenesis. Inhibition of angiogenesis is a new cancer treatment strategy that is now widely investigated clinically. Researchers have begun to search for objective measures that indicate pharmacologic responses to antiangiogenic drugs. Therefore, there is a great interest in techniques to visualize angiogenesis in growing tumors noninvasively. Several markers have been described that are preferentially expressed on newly formed blood vessels in tumors (alpha(v)beta(3) integrin, vascular endothelial growth factor, and its receptor, prostate-specific membrane antigen) and in the extracellular matrix surrounding newly formed blood vessels (extra domain B of fibronectin, Tenascin-C, matrix metalloproteinases, and Robo-4). Several ligands targeting these markers have been tested as a radiotracer for imaging angiogenesis in tumors. The potential of some of these tracers, such as radiolabeled cyclic RGD peptides and radiolabeled anti-PSMA antibodies, has already been tested in cancer patients, while for markers such as Robo-4, the ligand has not yet been identified. In this review, an overview on the currently used nuclear imaging probes for noninvasive visualization of tumor angiogenesis is given.

Development and Application of Peptide-Based Radiopharmaceuticals

During the past decade, radiolabeled receptor-binding peptides have emerged as an important class of radiopharmaceuticals for tumor diagnosis and therapy. The specific receptor binding property of the ligand can be exploited by labeling the ligand with a radionuclide and using the radiolabeled ligand as a vehicle to guide the radioactivity to the tissues expressing a particular receptor. The concept of using radiolabeled receptor binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. Receptor binding peptides labeled with gamma emitters ((123)I, (111)In, (99m)Tc) can visualize receptor-expressing tissues, a technique referred to as peptide-receptor radionuclide imaging (PRRI). In addition, labeled with beta emitters ((131)I, (90)Y, (188)Re, (177)Lu) these peptides have the potential to irradiate receptor-expressing tissues, an approach referred to as peptide-receptor radionuclide therapy (PRRT). The first and most successful imaging agent to date is the somatostatin analog octreotide. It is used for somatostatin receptor scintigraphy and PRRT of neuroendocrine tumors. Other peptides such as Minigastrin, GLP-1, CCK, bombesin, substance P, neurotensin, and RGD peptides are currently under development or undergoing clinical trials. In this review, an overview of the criteria of peptide ligand development, the selection of radioisotopes, labeling methods, and chemical aspects of radiopeptide synthesis is given. In addition, the current state of clinical use of radiopeptides for diagnosis and therapy of tumors is discussed.

Imaging integrin alpha-v-beta-3 expression in tumors with an 18F-labeled dimeric RGD peptide.

Integrin αv β3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine-glycine-aspartatic acid (RGD) peptide and integrin αv β3 , but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of (18)F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with (18)F in a simple one-pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA-E-[c(RGDfK)]2 labeled with (18)F at a specific activity of 1.8 MBq nmol(-1) and a radiochemical purity of 100% could be achieved. The logP value of (18)F-labeled NODAGA-E-[c(RGDfK)]2 was -4.26 ± 0.02. In biodistribution studies, (18)F-NODAGA-E-[c(RGDfK)]2 cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g(-1)) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of (18)F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID g(-1), 2 h p.i.) was significantly lower than that of reference compounds (68) Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID g(-1) ; p <0.001) and (111) In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID g(-1) ; p < 0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with (18)F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g(-1)). The αv β3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with (18)F-labeled NODAGA-E-[c(RGDfK)]2 . In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with (18)F using a direct aqueous, one-pot method and it accumulated specifically in αv β3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET.

Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation

Functional synergism and inhibitory effects of chemokine heterodimers can be selectively targeted by specific peptides in models of inflammation. Hampering heterodimers interrupts inflammation Inflammation is dependent on the recruitment of cells responding to chemokines. Von Hundelshausen et al. cataloged how human chemokines interact with each other and found that certain kinds of chemokine pairs can activate or inhibit receptor signaling. These chemokine heterodimers were shown to be active in mouse models of acute and chronic inflammation, which were ameliorated by treatment with a peptide designed to disrupt the chemokine pairing. Patients suffering from inflammatory conditions such as atherosclerosis could benefit from these kinds of therapeutics. Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.

Nuclear medicine imaging and therapy of neuroendocrine tumours

Radiolabelled peptides are used for specific targeting of receptors (over-)expressed by tumour cells. Dependent on the kind of labelling and the radionuclide used, these compounds may be utilised for imaging or for therapy. A concise overview is provided on basic principles of designing and developing radiopeptides for these applications. Furthermore, clinical application of these compounds for imaging and therapy is described. Advantages of the method compared to other techniques (such as the use of radiolabelled antibodies or antibody fragments) are discussed as well as pitfalls and limitations.