Urokinase-type plasminogen activator receptor (UPAR)-PET / CT in neuroendocrine tumors

Abstract

The present invention relates to a positron emitting imaging agent for prognostic use in patients with neuroendocrine tumors (NENs) undergoing PET imaging for cancer, said imaging agent comprising a uPAR binding peptide linked to the radionuclide 68Ga or 64Cu via the chelators NOTA or DOTA. The present invention also relates to a composition comprising a radiopharmaceutical for use in treating or alleviating neuroendocrine tumors (NENs) in a subject, said radiopharmaceutical comprising a radionuclide and a uPAR binding peptide. Figure 4A will be published together with the abstract.

Description

[Technical field]

[0001] The present invention relates to a positron emitting imaging agent for prognostic use in patients with neuroendocrine tumors (NEN) undergoing PET imaging of cancer, the imaging agent comprising a uPAR binding peptide bound to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA. The present invention also relates to a composition comprising a radiopharmaceutical for use in treating or alleviating a neuroendocrine tumor (NEN) in a subject, the radiopharmaceutical comprising a radionuclide and a uPAR binding peptide. Finally, the present invention relates to the use of the imaging agent as a companion diagnostic to the therapeutic radiopharmaceutical. [Background technology]

[0002] Neuroendocrine tumors (NENs) originate from neuroendocrine cells and are found primarily in the gastrointestinal tract, pancreas, and lungs. Patients diagnosed with NENs have a variable clinical course, ranging from indolent to highly advanced. The origin of the primary tumor, the presence of metastases, tumor morphology, and proliferative activity (e.g., Ki-67) are known prognostic factors. Patients are classified into NET G1 (Ki67<3%), NET G2 (Ki67 3-20%), NET G3 (Ki67>20% and well-differentiated), and NEC (Ki67>20% and poorly differentiated) according to the WHO classification (1). Noninvasive monitoring of tumor markers may be useful to further improve prognosis and guide treatment. PET is ideal for achieving this goal, as it allows the application of various specific radioactive tracers to noninvasively visualize the whole-body expression of tumor markers. In particular, in NENs, the expression of somatostatin receptors (e.g., 64 Cu-DOTATATE or 68 Ga-DOTATATE) and glucose metabolism ( 18 Radioactive tracers, such as F-FDG, are useful for diagnosis, prognosis, and treatment selection (2, 3). In addition, 177Peptide receptor radionuclide therapy (PRRT) using Lu-DOTATATE is approved for low-grade NENs, but its application may be limited in high-grade NENs due to low somatostatin receptor expression.

[0003] Therefore, improved prognostic tools for NEN would be advantageous, and in particular more efficient and / or reliable treatments for NEN subtypes would be advantageous. Summary of the Invention

[0004] The urokinase plasminogen activator receptor (uPAR) is a promising diagnostic and prognostic biomarker and therapeutic target that has been extensively studied in several cancer entities (4), but its relevance to NENs has not been investigated so far. uPAR is anchored to the cell membrane at the surface and localizes the proteolytic activity of its ligand, urokinase plasminogen activator (uPA). In normal tissues, uPAR expression is limited, whereas in cancer, uPAR expression is upregulated. In addition to uPA, uPAR also interacts with other proteins, including the integrin family of membrane proteins. Overall, uPAR is involved in promoting cell proliferation, motility, invasion, proteolysis, and angiogenesis (4-6). Due to its important role in cancer, PET radiotracers employing high affinity antagonists for uPAR have been developed. 68 Ga-NOTA-AE105 was developed by our group (7-9). Its safety and biodistribution were investigated in phase I studies, and its efficacy in primary and metastatic tumors was evaluated. 68 Accumulation of Ga-NOTA-AE105 was confirmed and correlated with uPAR expression in resected tumor samples (7). 68We have reported that uPAR PET using Ga-NOTA-AE105 can distinguish between low-risk and intermediate-risk profiles in prostate cancer (10). Furthermore, we have previously shown that PRRT targeting uPAR has high efficacy in preclinical studies of prostate and colorectal cancer (11, 12). Thus, uPAR, a marker of malignant disease, may be upregulated in aggressive NENs, providing a target for PRRT in these patients.

[0005] The purpose of this study in patients with NEN was to evaluate the uPAR-PET ( 68 The objective of this study was to evaluate tumor uptake and clinical outcomes using Ga-NOTA-AE105 (illustrated in PET / CT). 68 Because uPAR-PET / CT using Ga-NOTA-AE105 showed accumulation in NENs, it was hypothesized that uptake of the uPAR tracer would be associated with progression-free survival (PFS) and overall survival (OS) (see Example 5).

[0006] Surprisingly, uPAR-PET proved to be a more powerful prognostic tool (higher hazard ratio (HR)) (Figures 4A-4B) than PET-like instruments commonly used for neuroendocrine tumors, such as FDG-PET (see references in Figure 6) and somatostatin receptor imaging (e.g. Cu-DOTATATE (see references in Figure 7)). The latter was not significant with regard to OS.

[0007] Therefore, an object of the present invention is to improve prognostic tools for NENs.

[0008] In particular, it is an object of the present invention to improve treatment protocols for patients suffering from NENs.

[0009] Thus, one aspect of the present invention relates to a positron emitting imaging agent for use in prognostic diagnosis of patients with neuroendocrine tumors (NENs) undergoing PET imaging of their cancer; said imaging agent comprises a uPAR binding peptide bound to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA; and The uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof.

[0010] In one embodiment of the invention, the imaging agent comprises a uPAR binding peptide bound to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA, the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; The uPAR binding mutant is selected from the group consisting of the following mutants: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and ·(Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile); selected from the group consisting of; A quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TTR) exceeding a threshold level indicates poor prognosis for recurrence-free survival (RFS) and / or overall survival (OS); and A quantitative level of the SUVmax and / or SUVmean and / or the target-to-liver ratio (TTR) lower than a threshold level suggests a good prognosis for recurrence-free survival (RFS) and / or overall survival (OS); The present invention relates to a positron-emitting imaging agent for use in prognostic diagnosis of recurrence-free survival (RFS) and / or overall survival (OS) in patients with neuroendocrine tumors (NEN) who have undergone PET imaging of their cancer.

[0011] The two uPAR-PET tracers, 64Cu-DOTA-AE105 and 68Ga-NOTA-AE105, share the same binding moiety, the peptide AE105. Furthermore, they have both been tested in both humans and animals (see references (7), (28) and WO 2014 / 086364(A1)) and have been demonstrated to be similarly taken up in breast, bladder and prostate cancers. Thus, without being bound by theory, it is believed that the PET tracers - 64Cu or 68Ga as radionuclides; DOTA or NOTA as chelating agents; and a uPAR-binding peptide according to the invention; and is thought to have a similar effect on NEN patients. Another aspect of the invention relates to a positron emitting imaging agent for use in prognostic diagnosis in patients with neuroendocrine tumors (NENs) undergoing PET imaging for cancer; The imaging agent comprises a uPAR-binding peptide bound to the radionuclide 68Ga via the chelator NOTA; and The uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) (AE105) or a uPAR-binding variant thereof. In the Examples, such imaging agents are tested.

[0012] Yet another aspect of the present invention provides a method of in vivo PET imaging for assessing prognosis of a neuroendocrine tumor (NEN) in a patient, the method comprising: a) providing a subject who has previously been administered an imaging agent according to the invention; b) detecting radioactive emissions from the radioisotope contained in the imaging agent administered in step a) by in vivo PET imaging; c) generating an image representative of the location and / or amount of said radioactive emissions; d) determining the distribution and extent of uPAR expression in said subject, said expression being correlated with said signal emitted by said in vivo imaging agent; and e) UVmax and / or SUVmean and / or TLR quantitative levels exceeding the threshold levels indicate poor prognosis for recurrence-free survival (RFS) and / or overall survival (OS); and - Quantitative levels of SUVmax and / or SUVmean and / or TTR lower than the threshold levels indicate a favorable prognosis for recurrence-free survival (RFS) and / or overall survival (OS); comparing the distribution and extent of the resulting uPAR expression to a threshold level; Includes.

[0013] Yet another aspect of the invention is that the imaging agent comprises a uPAR binding peptide conjugated to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA; the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; Use of a positron emitting imaging agent according to the invention in the prognosis of patients with neuroendocrine tumors (NEN) undergoing in vivo PET imaging of uPAR expressing tumors.

[0014] Preferably, the imaging agent comprises a uPAR binding peptide conjugated to the radionuclide 68Ga via the chelator NOTA.

[0015] As outlined above, the methods of the present invention and the compositions used in the present invention can also be used as companion diagnostics. Thus, a further aspect of the present invention is The imaging agent comprises a uPAR binding peptide bound to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA; and the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; If the quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) exceeds a threshold level, it is suggested that the uPAR binding drug (e.g., a uPAR binding drug that is a radiopharmaceutical) is effective against the NEN; and A quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) lower than a threshold level indicates that the uPAR-binding drug (e.g., a uPAR-binding drug that is a radiopharmaceutical) is not effective against said NEN; This paper relates to a positron-emitting imaging agent for use as a companion diagnostic for PET imaging of cancer in patients with neuroendocrine tumors (NEN).

[0016] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: the imaging agent comprises a uPAR binding peptide bound to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA; the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; if the quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) exceeds a threshold level, it is suggested that a uPAR-binding drug, such as a uPAR-binding radiopharmaceutical, is effective against said NEN; A quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) lower than a threshold level indicates that a uPAR-binding drug, such as a uPAR-binding radiopharmaceutical, is not effective against the NEN; The uPAR binding mutant is the following mutant: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), ·Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and ·(Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile); Selected from the group consisting of: The present invention relates to an electron-emitting imaging agent for use in companion diagnostic PET imaging of cancer in patients with neuroendocrine tumors (NEN).

[0017] Further, one aspect of the present invention is the radiopharmaceutical comprises a radionuclide and a uPAR binding peptide; the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; The subject has been diagnosed with a uPAR-expressing neuroendocrine tumor (NEN); and said uPAR expression is above a predetermined threshold level; said threshold level being determined using a positron emitting imaging agent according to the present invention; The present invention relates to a composition comprising a radiopharmaceutical for use in treating or alleviating a neuroendocrine tumor (NEN) in a subject.

[0018] Further, one aspect of the present invention is The radiopharmaceutical comprises a radionuclide and a uPAR binding peptide; and the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; and The uPAR binding mutant is the following mutant: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and ·(Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile); selected from the group consisting of; the subject has been diagnosed with a uPAR-expressing neuroendocrine tumor (NEN), said uPAR expression being above a predefined threshold level; said threshold level being determined using a positron emitting imaging agent according to the present invention; The present invention relates to a composition comprising a radiopharmaceutical for use in treating or alleviating a neuroendocrine tumor (NEN) in a subject. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 shows a representative example of uPAR PET / CT imaging. Left column: CT. Right column: uPAR PET. Four patients (#AD) with high- and low-grade NEN were examined. The values ​​at the top of the individual scale bars correspond to the tumor SUVmax values. Patient #A: Bronchopulmonary NEC (Ki67: 24%). Patient #B: Orbital metastasis from small intestinal NET G2 (Ki-67: 5%). Patient #C: Large liver metastasis from pancreatic NET G2 (Ki-67: 5%). Patient #D: Large intramuscular metastasis from primary colonic NEC (Ki-67: 90%). [Figure 2A] Figure 2 shows Kaplan-Meier plots of (A) progression-free survival (PFS) and (B) overall survival using 68Ga-NOTA-AE105 uPAR PET. uPAR target-to-liver ratios were dichotomized at the median (TLR: 2.47). [Figure 2B]Figure 2 shows Kaplan-Meier plots of (A) progression-free survival (PFS) and (B) overall survival using 68Ga-NOTA-AE105 uPAR PET. uPAR target-to-liver ratios were dichotomized at the median (TLR: 2.47). [Diagram 3] Figure 3 shows the "Cutoff Finder" output, which represents the hazard ratio and 95% confidence interval (HR with 95% CI) when uPAR TLR is dichotomized at any possible cut point (x-axis). The solid vertical lines for (A) recurrence-free survival (PFS) and (B) overall survival using 68Ga-NOTA-AE105 uPAR PET are placed on the median uPAR TLR (2.47). [Figure 4A] Figure 4 shows Kaplan-Meier plots of (A) progression-free survival (PFS) and (B) overall survival using 68Ga-NOTA-AE105 uPAR PET. The uPAR TLR is dichotomized at 1.32. [Figure 4B] Figure 4 shows Kaplan-Meier plots of (A) progression-free survival (PFS) and (B) overall survival using 68Ga-NOTA-AE105 uPAR PET. The uPAR TLR is dichotomized at 1.32. [Diagram 5] FIG. 5 shows cutoff finders for SUVmax for (A) PFS and (B) OS. [Figure 6] 6 is a diagram showing the Kaplan-Meier plot of FDG-PET in patients with neuroendocrine tumors, (left) recurrence-free survival (PFS), and (right) overall survival (OS). Citation from Reference 3 (Binderup et al., J Nucl Med 2021). This is a reference figure and is not part of the present invention. [Figure 7] Figure 7 shows the Kaplan-Meier plot of 64Cu-DOTATATE in patients with neuroendocrine tumors, (left) progression-free survival (PFS), and (right) overall survival (OS). Cited from Reference 22 (Carlsen, et al. J Nucl Med. 2020). This is a reference figure and is not part of the invention.

[0020] The present invention will be described in further detail below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] definition Before describing the present invention in further detail, the following terms and conventions will first be defined.

[0022] Neuroendocrine tumors (NENs) "Neuroendocrine tumors" (NENs or NETs) originate from neuroendocrine cells and are found primarily in the gastrointestinal tract, pancreas, and lungs.

[0023] Although there are many types of NENs, most are treated as a group because the cells of these tumors share common characteristics, such as a similar appearance, the presence of specialized secretory zymogen granules, and often the production of biogenic amines and polypeptide hormones.

[0024] Ga68 Gallium-68.

[0025] 64Cu Copper-64.

[0026] 177Lu 177Lu is a low-energy beta emitter (maximum penetration depth in soft tissues is about 1.5 mm) that can induce cytotoxic effects in tumors but not in surrounding tissues, either by "cross-fire" effects from direct beta particles and Auger electrons, or by "bystander" effects. Therefore, 177Lu is considered the radionuclide of choice for the treatment of large as well as small tumor lesions and disseminated metastatic disease.

[0027] AE105 Ac-Asp-Cha-Phe-(D)Ser-(D)Arg-Tyr-Leu-Trp-Ser The peptides of the invention can be synthesized, for example, using standard solid phase peptide chemistry.

[0028] NOTA NOTA:2,2',2 ” -(1,4,7-Triazacyclononane-1,4,7-triyl l) Triacetic acid. NOTA is coupled with AE105 to form NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser). The chemical structure is shown below:

[0029] In one embodiment, the imaging agent is 68Ga-NOTA-AE105.

[0030] [ka]

[0031] DOTA DOTA (also called tetraxetane) has the formula (CH 2 CH 2 NCH 2 CO 2 H) 4 DOTA is an organic compound. The molecule consists of a central 12-membered tetraaza (i.e., containing four nitrogen atoms) ring. DOTA is used as a complexing agent, especially for lanthanide ions. The complexes have medical applications as imaging agents and cancer treatment drugs.

[0032] Suggested IUPAC name for DOTA 2,2',2 ” ,2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid. Hereafter, DOTA is shown as a complex with 68Ga and 64CU bound to AE105.

[0033] Recurrence-free survival (RFS) The term "recurrence-free survival" (RFS) is defined as a clinical endpoint defined as the time from diagnosis to any recurrence of disease in the locoregion (TN site) and / or distant metastasis (M site), with censoring and death from other causes recorded as disease-free survival (DFS), which is defined as RFS, but includes death from any cause as an event.

[0034] Overall survival (OS) The term "overall survival" (OS) is defined as the time from diagnosis to death from any cause. Follow-up period is defined as the time from referral for radiotherapy to first recurrence, death, or end of follow-up on January 1, 2021.

[0035] Threshold Level In the context of the present invention, the terms "threshold level", "reference level" or "cutoff" relate to a standard value for a quantity that can be compared with another value or characteristic.

[0036] In one embodiment of the present invention, threshold levels can be determined by examining uPAR levels by PET / CT in healthy subjects. One or more threshold levels can be calculated by applying various statistical measures such as cut-off value detection, multivariate analysis, etc.

[0037] See also Example 5 for determining cutoff values.

[0038] Based on these results, a cut-off value can be obtained that indicates the relationship between the detected level and the patients at risk, and the cut-off value can then be used to determine the uPAR level that corresponds to, for example, an increased risk of poor RFS or poor OS.

[0039] Risk assessment The present inventors have succeeded in developing a new method for predicting the prognosis of subjects with neuroendocrine tumors (NEN), such as RFS and / or OS. To determine whether a patient is at increased risk for a poor prognosis, a cut-off value (reference level) must be set. This cut-off value can be set individually for each laboratory, physician, or patient.

[0040] The cut-off levels can be set using a variety of methods, including multivariate statistical tests (such as partial least squares discriminant analysis (PLS-DA), random forests, support vector machines, etc.), percentiles, mean plus or minus standard deviation, median, fold change, etc.

[0041] Multivariate discriminant analysis and other risk assessments can be performed using free or commercially available computer statistical packages (SAS, SPSS, Matlab, R, etc.) or other statistical software packages or screening software known to those skilled in the art.

[0042] As will be apparent to those skilled in the art, in any of the above embodiments, changing the risk cutoff level may change the results of the discriminant analysis for each subject.

[0043] Statistics allow us to assess each level of significance. Common statistical tests applied to data sets include t-tests, f-tests, or more advanced data comparison tests or methods. Such tests or methods can be used to determine whether two or more samples are significantly different.

[0044] The significance can be determined by standard statistical methods known to those of skill in the art.

[0045] The reference level selected may vary depending on the mammal / subject to which the test is applied.

[0046] Preferably, the subject according to the present invention is a human.

[0047] The selected reference level can be modified as needed to change it to specificity or sensitivity as known in the art. The sensitivity and specificity are statistical indices that are widely used to describe and quantify how good and reliable a biomarker or diagnostic test is. The sensitivity evaluates how good a biomarker or diagnostic test is at detecting disease, and the specificity estimates how accurately an individual (i.e., a control, a disease-free patient) can be determined to be not at risk.

[0048] In describing the sensitivity and specificity, several terms are used: true positive (TP), true negative (TN), false negative (FN), and false positive (FP). A diagnostic test result is considered a TP if it proves the presence of a disease in a patient who has the disease. If the disease is not present in an individual (i.e., a control, a patient without the disease) and the diagnostic test confirms the absence of the disease, the test result is a TN. If the diagnostic test indicates the presence of a disease in an individual who does not have such a disease, the test result is a FP. Finally, if the diagnostic test indicates the absence of a disease in a patient who has the disease, the test result is an FN.

[0049] sensitivity As used herein, the sensitivity refers to a measure of the proportion of subjects who are correctly identified as actually positive, i.e., the proportion of subjects at risk of a poor prognosis above the normal value among those with a poor prognosis above the normal value.

[0050] The sensitivity of the test can usually be described as the proportion of true positives relative to the total number of patients with the disease of interest, i.e., the risk of poor prognosis above normal. All patients with the disease of interest are the sum of the true positive (TP) patients (detected) and the false negative (FN) patients (not detected).

[0051] specificity In the present specification, the specificity refers to a measure of the proportion of correctly identified negatives, i.e., the proportion of mammals that are not at increased risk of poor prognosis and are identified as not at risk for poor prognosis above the normal range. An ideal diagnostic test would be one that has a specificity of 100%, i.e., only subjects at risk for poor prognosis above the normal range are detected, i.e., there are no false positive patients, and a sensitivity of 100%, i.e., all subjects at risk for poor prognosis above the normal range are detected, i.e., there are no false negative patients.

[0052] Typically with any test there is a trade-off between each measure. For example, a manufacturing site checking for defects may be willing to risk discarding some parts that function properly (low specificity) in order to increase the chances of identifying nearly all defective parts (high sensitivity). This trade-off can be represented graphically using an ROC curve.

[0053] The selection of the specificity determines the proportion of false positive cases that is acceptable for a particular study / population and for a particular institution. By lowering the specificity, the sensitivity increases.

[0054] As is generally understood by those skilled in the art, prognostic screening methods are a decision-making process, and therefore the specificity and sensitivity chosen will vary depending on what a particular institution / clinician considers to be the optimal outcome.

[0055] SUVmax In the present context, the term "SUVmax" refers to the "maximum standardized uptake" (SUVmax), which is commonly used when measuring uPAR and FDG uptake by malignant tissues. The increased uptake is due to increased uptake or binding of the tracer to cancer cells, which can be imaged and quantified using PET.

[0056] SUVmean In the present context, the term "SUVmean" refers to the mean standardized uptake.

[0057] Target-to-liver ratio (TLR) TLR is defined as the ratio of lesion SUVmax / normal liver SUVmean. A positive lesion is evident when the lesion SUVmax / normal liver SUVmean is greater than 2.

[0058] Companion Diagnostics In this context, a "companion diagnostic" is a diagnostic test used as a companion to a therapeutic drug, the applicability of which to a particular person being determined.

[0059] Companion diagnostics may be developed in conjunction with drugs to help select or exclude patient groups for treatment with a particular drug based on biological characteristics that determine responders and non-responders to the treatment. The companion diagnostics are developed based on companion biomarkers, i.e., biomarkers that help prospectively predict expected response or severe toxicity.

[0060] Imaging agents for prognostic assessment in patients with neuroendocrine tumors (NENs) undergoing PET imaging Improving the prognosis of patients with neuroendocrine tumors (NENs) is important, for example, not only to determine whether radiation therapy is necessary, but also to determine the appropriate radiation therapy. Therefore, improving companion diagnostics is also important. One aspect of the present invention is to provide a method for the treatment of neuroendocrine tumors (NENs) by using a combination of a genomic DNA sequence and a genomic DNA sequence. The imaging agent comprises a uPAR binding peptide bound to the radionuclide 68Ga via the chelator NOTA; and The uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; This paper concerns positron-emitting imaging agents for use in prognostic assessment in patients with neuroendocrine tumors (NENs) undergoing PET imaging for cancer.

[0061] As outlined in the examples, the imaging agents of the present invention are powerful prognostic markers for NEN patients. Moreover, optimal cut-off values ​​have been identified. Furthermore, such markers are suitable as companion diagnostics, since they allow the identification of patients who are sensitive to radiotherapy using radionuclides conjugated to uPAR-binding peptides.

[0062] The different uPAR-binding variants of said uPAR-binding peptides, including AE105, are disclosed in WO 2014 / 086364 A1. Thus, in one embodiment, said uPAR-binding variant is selected from the group consisting of: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and (Asp)-([β]-Cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile).

[0063] The uPAR binding portion of the imaging agent is preferably that known as AE 105. Thus, in one embodiment, the peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser).

[0064] In other embodiments, the C-terminus is a carboxylic acid or an amide. In yet other embodiments, the imaging agent has the formula:

[0065] [ka] It is expressed as:

[0066] The PET scan may be combined with other scan types to further improve the localization of the cancer. In one embodiment, prognosis includes a PET / CT scan and / or a PET / MR scan. In the examples, PET / CT is used.

[0067] The prognosis may be further defined. In one embodiment, the prognosis is a prognosis for recurrence-free survival (RFS) and / or overall survival (OS). In the examples, RFS and OS are evaluated.

[0068] Varying amounts (MBq) of imaging agent may be used, as well as varying times before scanning after administration of the imaging agent. Thus, in one embodiment, the imaging agent is administered at a dose of 10-500MBq, followed by a PET scan 10 minutes to 24 hours after administration of the imaging agent, quantified using SUVmax and / or SUVmean and / or TLR.

[0069] In other embodiments, the imaging agent is administered at a dose of 20-400MBq, for example 50-400MBq, for example 70-300MBq, for example 100-300MBq, or for example 100-300MBq. Preferably 150-250MBq, more preferably 170-230MBq. Thus, in a preferred embodiment, the imaging agent is administered at a dose of 70-300MBq, preferably 150-250MBq. In the examples, about 200MBq was used, and PET imaging was performed with a Siemens Biograph at a sensitivity of 128mCT. However, other devices may be used with higher sensitivity and allow the amount (MBq) of the imaging agent to be reduced.

[0070] In one embodiment, a sufficient amount of the imaging agent is administered to allow PET imaging with sufficient radioactivity for imaging.

[0071] In the case of 68Ga, the half-life is about 1 hour (68 minutes), so the time before PET scanning is 10 minutes to 5 hours, preferably 20 minutes to 3 hours. In the case of 64Cu, the half-life is about 12.7 hours, so the time before PET scanning is 20 minutes to 24 hours.

[0072] In a related embodiment, the PET scan is performed 20 minutes to 10 hours, such as 20 minutes to 5 hours, such as 30 minutes to 3 hours, after administration of the imaging agent.

[0073] In yet another embodiment, the imaging agent of the present invention is present in a pharmaceutical composition comprising said imaging agent and one or more pharma- ceutical acceptable adjuvants, excipients and / or diluents.

[0074] A threshold level (cut-off value / reference level) may be included so that a prognosis can be made. In one embodiment, the imaging agent used according to the invention is A quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TTR) exceeding a threshold level indicates poor prognosis for recurrence-free survival (RFS) and / or overall survival (OS); and A quantitative level of the SUVmax and / or SUVmean and / or the target-to-liver ratio (TTR) lower than a threshold level suggests a good prognosis for recurrence-free period (RFS) and / or overall survival (OS).

[0075] In Example 5, the identified optimal threshold levels (cut-off values) are calculated.

[0076] In yet another embodiment, the target-to-liver ratio (TLR) threshold level is in the range of 1.1 to 4, such as in the range of 1.3 to 4, such as in the range of 2 to 3. Median dichotomized uPAR TLR was significantly associated with PFS and OS, with patients with high uPAR expression having a significantly worse prognosis (Example 5, Tables 4 and 5).

[0077] In yet another embodiment, the threshold level of the target-to-liver ratio (TLR) is in the range of 1.1 to 2, such as in the range of 1.1 to 1.5, such as in the range of 1.2 to 1.4. A lower cutoff value for the TLR (1.32) allows the identification of a smaller patient population (n=10) with no or very low risk of death or progression (Example 5, Figure 4).

[0078] In another embodiment, the threshold level of SUVmax and / or SUVmean is in the range of 1 to 4, such as in the range of 2 to 4, preferably in the range of 2 to 3, such as in the range of 2 to 2.5. As outlined in Example 5 (Figures 5A,B), an SUV threshold level can also be established that is not necessarily superior to the TLR threshold.

[0079] In yet another embodiment, the threshold level is determined by a cutoff detection method to obtain a partition in a Kaplan-Meier plot (log-rank test) and a corresponding hazard ratio (HR).

[0080] In one embodiment, neuroendocrine tumors (NENs) are classified as NET G1, NET G2, NET G3, or NEC according to the WHO classification (1). Example 3 shows that uPAR PET can distinguish between low-grade (NET G1 / G2) and high-grade NENs (NET G3 and NEC).

[0081] Of course, it should be noted that embodiments of other aspects of the invention are also applicable to this aspect.

[0082] In vivo imaging method using PET imaging to evaluate the prognosis of neuroendocrine tumors (NEN) Another aspect of the invention relates to a method of in vivo imaging, e.g. by PET imaging to assess the prognosis of a patient suffering from a neuroendocrine tumor (NEN), said method comprising: a) providing a subject to whom an imaging agent according to the invention has previously been administered; b) detecting radioactive emissions from the radioisotope in the imaging agent administered in step a) by in vivo PET imaging; c) generating an image representative of the location and / or amount of said radioactive emissions; d) determining the distribution and extent of uPAR expression in said subject, said expression correlating with said signal emitted by said in vivo imaging agent; and e) A quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TTR) exceeding a threshold level indicates poor prognosis for recurrence-free survival (RFS) and / or overall survival (OS); and if the quantitative level of the SUVmax and / or SUVmean and / or the target-to-liver ratio (TTR) is lower than a threshold level, it suggests a good recurrence-free survival (RFS) and / or overall survival (OS) of the prognosis; comparing the distribution and extent of the determined uPAR expression to a threshold level; Includes.

[0083] In one embodiment, if a favorable prognosis for recurrence-free survival (RFS) or favorable prognosis for overall survival (OS) is indicated, a de-escalation of the treatment regimen is planned for the subject, avoiding the use of unnecessarily high toxicity, and / or a risk-stratified follow-up schedule is established. As used herein, a de-escalated treatment protocol is one that is considered to be less toxic / harmful to the patient compared to patients with a poor prognosis for recurrence-free survival (RFS).

[0084] In one embodiment, the de-escalating treatment is the withholding or reduction of radiation therapy.

[0085] In another embodiment, the subject will be treated with radiation therapy if a poor prognosis for recurrence-free survival (RFS) or poor prognosis for overall survival (OS) is indicated.

[0086] In yet another embodiment, the radiation therapy is carried out using a radiopharmaceutical comprising a radionuclide and a uPAR binding peptide, such as conjugated via a chelator.

[0087] In yet another embodiment, the treatment is any anti-cancer therapy.

[0088] In a further embodiment, the levels expressed as SUVmax, SUVmean, and / or TLR determine whether a patient is a candidate for targeted radionuclide therapy.

[0089] In one embodiment, the method forms the basis for a companion diagnostic for targeted radionuclide therapy. Thus, in such an embodiment: If the quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) exceeds a threshold level, it is suggested that the uPAR-binding agent, e.g., a uPAR-binding radiopharmaceutical, is effective against said NEN; and If the quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) is lower than a threshold level, it is suggested that the uPAR-binding agent, e.g., a uPAR-binding radiopharmaceutical, is not effective against said NEN.

[0090] Preferably, said agent is a uPAR binding radiopharmaceutical, more preferably said radiopharmaceutical is 177Lu-DOTA-AE105.

[0091] Use of positron-emitting imaging agents in the prognosis of patients with neuroendocrine tumors (NENs) using in vivo PET imaging A further aspect of the present invention is the imaging agent comprises a uPAR binding peptide conjugated to the radionuclide 68Ga via the chelator NOTA; the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; The use of a positron emitting imaging agent according to the invention in the prognosis of patients with neuroendocrine tumors (NEN) using in vivo PET imaging of uPAR expressing tumors.

[0092] In some alternative embodiments of this embodiment, the imaging agent comprises a uPAR binding peptide bound to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA.

[0093] Again, in one embodiment, said uPAR binding mutants thereof are the following mutants: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and ·(Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile); is selected from the group consisting of:

[0094] Companion Diagnostics As outlined above, the method of the invention and the composition used according to the invention can also be used as a companion diagnostic. Thus, a further aspect of the invention is the imaging agent comprises a uPAR binding peptide conjugated to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA; the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; if the SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) quantification level exceeds a threshold level, it is suggested that a uPAR-binding drug, such as a uPAR-binding radiopharmaceutical, is effective against said NEN; If the SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) quantification levels are lower than the threshold levels, it is suggested that the uPAR-binding drug, such as the uPAR-binding radiopharmaceutical, is not effective against the NEN; This paper concerns a positron-emitting imaging agent for use as a companion diagnostic for patients with neuroendocrine tumors (NENs) undergoing PET imaging for cancer.

[0095] In a preferred embodiment, the imaging agent is 68Ga-NOTA-AE105. The imaging agent is used in the examples.

[0096] In another preferred embodiment, the drug is a radiopharmaceutical. In a further preferred embodiment, the uPAR binding radiopharmaceutical is 177Lu-DOTA-AE105.

[0097] Again, it should be noted that embodiments of other aspects of the invention are also applicable to this aspect.

[0098] Radiopharmaceuticals for use in treating or alleviating a subject's neuroendocrine tumor (NEN) As outlined above, the imaging agents may be used as companion diagnostics to distinguish subtypes of NENs that express specific up- or down-regulated surface proteins, and the up-regulated surface proteins may be used to target radiopharmaceuticals to the cancer.

[0099] Yet another aspect of the present invention is the radiopharmaceutical comprises a radionuclide and a uPAR binding peptide; said uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; and said subject has been diagnosed with a uPAR-expressing neuroendocrine tumor (NEN), said uPAR expression being above a predetermined threshold level: determining said threshold level using a positron emitting imaging agent according to the present invention; The present invention relates to a composition comprising a radiopharmaceutical for use in treating or alleviating a neuroendocrine tumor (NEN) in a subject.

[0100] Preferably, the radionuclide and peptide are linked via a chelating agent such as DOTA or NOTA.

[0101] In one embodiment, the radionuclide is for targeted radionuclide therapy (alpha, beta-emitter or Auger) and is selected from the group of isotopes consisting of 67Cu, 177Lu, 89Sr, 90Y, 117mSn, 131I, 153Sm, 166Ho, 186Re, 188Re, 211At, 212Pb, 212Bi, 213Bi, 223Ra, 224Ra, 225Ac, 227Th, preferably 177Lu, 67Cu, 90Y, 211At, 225Ac, and 227Th, more preferably 177Lu.

[0102] In another embodiment, the radiopharmaceutical is conjugated to the uPAR binding peptide via a chelator, such as, for example, DOTA, CB-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A, TETA, TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, SarAr, SarAr-NCS, diamSar, AmBaSar, BaBaSar, ATSM, CB-TE1A1P and CB-TE2P, NOTA, NETA, TACN-TM, NODAGA, TRAP, AAZTA, DATA, H2dedpa, CP256, PCTA, THP, DTPA, 1B4M-DTPA, CHX-A"-DTPA, TRAP(PRP9), NOPO, DFO The chelator is selected from the group consisting of HOPO, H6phospa, PCTA, H2dedpa, H4octapa, H2azapa, H5decapa, HBED, HBED-cc, SHBED, BPCA, CP256, HEHA, PEPA, and RESCA1; preferably selected from DOTA, NOTA, CB-TE2A, NODAGA, DFO, HBED, and HBED-cc, and more preferably, the chelator is DOTA or NOTA.

[0103] In one embodiment, said threshold level of target-to-liver ratio (TLR) is in the range of 1.1-4, such as in the range of 1.3-4, such as in the range of 2-4 or 2-3. In another embodiment, the threshold level of the target-to-liver ratio (TLR) is in the range of 1.1 to 2, such as in the range of 1.1 to 1.5, such as in the range of 1.2 to 1.4.

[0104] In yet another embodiment, the radiopharmaceutical is 177Lu-DOTA-AE105.

[0105] In yet another embodiment, the radiopharmaceutical has the formula:

[0106] [ka] Has.

[0107] It should be noted that embodiments of other aspects of the invention are also applicable to this aspect.

[0108] Additional Aspects of the Invention A further aspect of the present invention is the imaging agent comprises a uPAR binding peptide conjugated to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA; The uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; This paper relates to a positron-emitting imaging agent for use in prognostic assessment of patients with neuroendocrine tumors (NENs) undergoing PET imaging of their cancer.

[0109] Again, in one embodiment, the uPAR binding mutant is the following mutant: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and ·(Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile); is selected from the group consisting of:

[0110] In one embodiment, the imaging agent comprises 68Ga-NOTA and a uPAR binding peptide as outlined herein.

[0111] In one embodiment, the imaging agent comprises 68Ga-DOTA and a uPAR binding peptide as outlined in the present invention. In one embodiment, the imaging agent has the following formula:

[0112] [ka] has.

[0113] In one embodiment, the imaging agent comprises 64Cu-NOTA and a uPAR binding peptide as outlined in the present invention. In one embodiment, the imaging agent has the following formula:

[0114] [ka] has.

[0115] In one embodiment, the imaging agent comprises 64Cu-DOTA and a uPAR binding peptide as outlined in the present invention. In one embodiment, the imaging agent has the following formula:

[0116] [ka] has.

[0117] The different variants of the radionuclide-chelator-uPAR binding peptide (including AE105) are also disclosed in WO 2014 / 086364(A1), and the compounds are incorporated herein by reference. The two uPAR-PET tracers 64Cu-DOTA-AE105 and 68Ga-NOTA-AE105 share the same binding moiety, the peptide AE105. Moreover, both have been tested in both humans and animals (see references (7), (28) and WO 2014 / 086364(A1)), and have been demonstrated to be similarly taken up in breast, bladder and prostate cancers. Therefore, without being bound by theory, it is believed that the two PET tracers will work similarly in NEN patients.

[0118] It should be noted that embodiments and features described in the context of one aspect of the invention may also be applied to other aspects of the invention.

[0119] All patent and non-patent references cited herein are hereby incorporated by reference in their entirety.

[0120] The invention is further illustrated by the following non-limiting examples.

[0121] Working Example Example 1 - Materials and Methods patient Patients with histologically confirmed NEN were enrolled between November 17, 2017 and June 29, 2020 at the Department of Endocrinology (managed low-grade NEN, Ki67 ≤ 20%) and Department of Oncology (managed high-grade NEN, Ki67 > 20%), Rigshospitalet, Copenhagen University Hospital, Denmark. Rigshospitalet is a renowned neuroendocrine tumor center certified by the European Society for Neuroendocrine Tumors. The study was performed in accordance with the Declaration of Helsinki and good clinical practice.

[0122] Eligible patients were aged 18 years or older, able to read and understand patient information in Danish, able to provide informed consent, diagnosed with any grade of gastroenteropancreatic or bronchopulmonary NEN, and with a WHO performance status of 0-2. 68 Patients were excluded if they had a history of allergic reactions to Ga-NOTA-AE105 and compounds of similar chemical or biological composition, or if they had bronchopulmonary NEN with the subtype small cell lung cancer. After signing a written informed consent, the patients were first 68 Ga-NOTA-AE105 PET / CT was performed.

[0123] Image Acquisition Data were acquired using a Biograph 128mCT PET / CT (Siemens Medical Solutions) with an axial field of view of 216 mm. Based on a previous Phase I study (7), approximately 200 MBq of 68Ga-NOTA-AE105 was administered intravenously and scanned 20 min later. Tracer generation was performed as previously described (7). Whole-body PET scans (from mid-orbit to mid-thigh) were performed for 4 min each at different bed positions. Attenuation- and scatter-corrected PET data were iteratively reconstructed using a 3D conventional Poisson expectation-maximization algorithm including point spread function and time-of-flight information using the TrueX algorithm (Siemens Medical Solutions); the settings were 2 iterations, 21 subsets, and a 2 mm Gaussian filter. Prior to the PET scan, a diagnostic CT scan was performed using a Care Dose 4D automatic exposure control system (Siemens Medical Solutions) with a slice thickness of 2 mm, 120 kV, and quality standard of 225 mAs. During arterial and venous phase CT, 75 mL of iodine-containing imaging agent (Optiray 300, Covidien) was administered using an automatic injection system.

[0124] Image Analysis An experienced nuclear medicine physician and an experienced radiologist worked together to analyze the PET / CT scans. The physicians were blinded to the patient data. Lesions were identified by CT and / or PET. SUV was calculated as decay-corrected measured activity concentration / (injected activity / body weight). In cases with multiple organ lesions, 68 Ga-NOTA-AE105 SUV max The lesion with the highest SUV value was recorded. If no uPAR-positive lesions could be identified but lesions were present on CT, the largest lesion on CT (based on viable tumor) was used as a guide for delineating on the PET scan. max Physiological liver uptake was assessed in normal liver tissue from all patients (preferably from the right side of the liver, avoiding major blood vessels). To measure and standardize uPAR expression within and between patients, the uPAR target-to-liver ratio (uPAR TLR) was used to measure and normalize lesion SUV max / Normal Liver SUVmean Lesions were defined as uPAR positive if ≧2.

[0125] Follow-up The patients were regularly examined at the renowned Rigshospitalet Neuroendocrine Oncology Center and underwent clinical examinations, blood samples, and imaging studies (CT, MR, ultrasound, PET / CT) at frequencies according to ENETS guidelines (13). Endpoint follow-up was performed on July 8, 2021. Conventional CT and / or magnetic resonance imaging were used to assess PFS according to the Response Evaluation Criteria in Solid Tumors, v.1.1 (14). PFS was calculated as the time from uPAR PET / CT to progression or death from any cause. Patients were censored at the time of last diagnostic imaging if no progression or death from any cause occurred during follow-up. OS was calculated as the time from uPAR PET / CT to death from any cause. All deaths except two were directly related to NENs, so disease-specific survival analyses were reserved. Patients who were alive at follow-up were censored at the date of follow-up, i.e., July 8, 2021.

[0126] statistics The sample size was determined based on previous studies of prognostic markers in patients with NEN (15,16), and a 1-year follow-up of 100 patients was sufficient to detect significant differences in PFS and OS between the groups (type I error risk 0.05, power 0.8). Taking into account dropouts, the number of subjects was set at 120 patients. Continuous variables were reported as mean (standard deviation (SD) or median with range. Kaplan-Meier analysis was used to estimate time to outcome (PFS and OS) and inverse Kaplan-Meier analysis was used to estimate median follow-up duration. Optimal cutoff values ​​for uPAR TLR (17) were determined using an application (Cutoff Finder). Univariate and multivariate Cox regression analyses of OS and PFS were performed with uPAR TLR and WHO grade as predictor variables. A p value <0.05 was considered statistically significant. Analyses were performed using R version 3.6.0 (R Foundation for Statistical Computing, Vienna, Austria).

[0127] Example 2 - Patients and image acquisition One hundred twenty patients were followed consecutively, of which 21 patients did not undergo uPAR PET / CT (disease worsening, n=5; consent withdrawal, n=5; did not meet inclusion criteria, n=2; died before uPAR PET / CT, n=4; could not perform uPAR PET / CT due to COVID-19 restrictions, n=5). Of the 99 patients scanned with uPAR PET / CT, 96 patients had evaluable disease (failed to perform uPAR PET / CT due to technical issues, n=1; no lesions seen on either CT or PET, n=2). Patient demographic data for the study cohort (n=96) are shown in Table 1. The majority of patients had small bowel NEN (64%, 61 / 96) and 90% (86 / 96) had metastatic disease. Additionally, the cohort was dominated by patients with high-grade disease, with 9% (9 / 96) having NET G3 and 16% (15 / 96) having NEC.

[0128] [Table 1] JPEG2025501716000009.jpg135170*Some patients received multiple treatments, so number of treatments exceeds number of patients. Data are shown as percentages in parentheses after numbers unless otherwise noted. Percentages have been rounded and may not add up to 100%. NET: neuroendocrine tumor. NEC: neuroendocrine carcinoma.

[0129] Patients received a median (range) of 194 (104-236) MBq 68 Ga-NOTA-AE105 and 17.2 (8.7-39.8) μg of AE105 were injected. The median (range) time from injection to PET scan was 22 (18-38) minutes. One patient experienced an adverse event (mild nausea), which was not reported. 68 All events were deemed unrelated to Ga-NOTA-AE105 injections. No serious adverse events were recorded.

[0130] Example 3 - Imaging analysis Tracer uptake in normal liver tissue was taken as the tumor uptake measure. Normal liver SUV mean The mean (SD) of uPAR-positive lesions was 1.50 (0.39). uPAR-positive lesions were observed in both low-grade (NET G1 / G2) and high-grade NEN (NET G3 and NEC) patients (Table 2). Representative examples of uPAR PET are shown in Figure 1.

[0131] [Table 2]

[0132] Data are numbers with percentages in parentheses. uPAR target-to-liver ratios were used to calculate lesion SUV max / Normal Liver SUV mean Lesions were defined as uPAR positive if ≥ 2. Among G3 patients, 8 of 9 had NET G3 and 10 of 15 had NEC uPAR PET positive.

[0133] conclusion Thus, uPAR PET can distinguish between low-grade (NET G1 / G2) and high-grade NEN (NET G3 and NEC).

[0134] Example 4 - Follow-up The median follow-up period after uPAR PET / CT was 28 months. During follow-up, 59 (62%) patients experienced progressive disease, with a median PFS of 17.3 months and 28 patients (30%) dying. Treatment of patients after uPAR PET / CT is shown in Table 3. Treatment with somatostatin analogues was the most common (77%, 74 / 96), with 28% (27 / 96) of all patients receiving peptide receptor radionuclide therapy during the follow-up period.

[0135] [Table 3]

[0136] Some patients received multiple treatments, so the number of treatments exceeds the number of patients. Data are numbers with percentages in parentheses.

[0137] Example 5 - Recurrence-free survival and overall survival Research Objective To determine progression-free survival (PFS) and overall survival (OS) based on uPAR imaging.

[0138] result uPAR TLR as a continuous variable was significantly associated with PFS with HR (95% CI) 1.27 (1.02-1.60), p=0.04.

[0139] For OS, uPAR TLR as a continuous marker reached borderline statistical significance with HR (95%CI) 1.37 (0.98-1.92), p=0.06. TLR was then dichotomized at the median (2.47) and Kaplan-Meier analysis was performed (Figure 2). OS was not reached in the low uPAR expression group (TLR<median) and 32.1 months (23.8, upper limit not reached) in the high uPAR expression group (TLR≥median). Median PFS was 22.1 months (14.7, upper limit not reached) in patients with low uPAR expression and 14.1 months (11.4-22.4) in patients with high uPAR expression. uPAR TLR dichotomized at the median was significantly associated with PFS and OS, with patients with high uPAR expression having a significantly worse prognosis (Tables 4 and 5).

[0140] [Table 4] *uPAR TLR dichotomized at the median (2.47). TLR: target-to-liver ratio. NET: neuroendocrine tumor. NEC: neuroendocrine carcinoma. HR: hazard ratio. CI: confidence interval.

[0141] [Table 5] *uPAR TLR dichotomized at the median (2.47). TLR: target-to-liver ratio. NET: neuroendocrine tumor. NEC: neuroendocrine carcinoma. HR: hazard ratio. CI: confidence interval.

[0142] Other cut-off values ​​were evaluated using the cut-off finder shown in Figures 3A and B. Using a lower cut-off value for TLR (1.32), we were able to identify a smaller group of patients (n = 10) with no or very low risk of death or progression (Figure 4). Patients with NET G3 and NEC had significantly worse PFS and OS compared with NET G1, whereas no differences were observed between NET G2 and NET G1 (Tables 4 and 5). In multivariate analysis including uPAR expression and WHO classification, both were significantly associated with PFS, but when WHO grade was considered, uPAR expression correlated with OS (P = 0.06) with borderline statistical significance.

[0143] For SUVmax, a cut-off value of 2–2.5 could be established, which showed a significant difference in prognosis (hazard ratio (HR) for recurrence-free survival (PFS) was 3–5) (Figure 5A). Similarly, for SUVmax, a cut-off value of 2.3 could be established, with an HR for overall survival (OS) of 4–8 (significance level 2.3) (Figure 5B). However, this identified only 5–6 patients as low risk. Thus, cut-off values ​​using TLR were superior to SUVmax in establishing PFS and OS.

[0144] conclusion When dichotomized at the median, uPAR TLR was significantly associated with PFS and OS, and patients with high uPAR expression had a significantly worse prognosis.

[0145] Furthermore, by using a lower TLR cutoff, a smaller group of patients at no or very low risk of death or progression could be identified.

[0146] Importantly, the cutoff values ​​established using TLR were superior to those established using SUVmax. In particular, for companion diagnostics of targeted radiotherapy, TLR is important because it reflects the effect of tumor on background radiation, i.e., the effect of tumor on normal tissue radiation (side effects).

[0147] Finally, as outlined in the Summary of the Invention, we surprisingly found that uPAR-PET is a more powerful prognostic tool (higher hazard ratio (HR)) (Figures 4A-4B) than PET-like instruments commonly used for neuroendocrine tumors, such as FDG-PET (see reference diagram in Figure 6) and somatostatin receptor imaging (e.g. 64Cu-DOTATATE (see reference diagram in Figure 7)). The latter was not significant with regard to OS.

[0148] Example 6 - Imaging analysis and comparison with somatostatin receptor imaging Tracer uptake in normal liver tissue was used as a measure of tumor uptake. Comparing uPAR positivity with published data in the literature on somatostatin receptor positivity (using this "positivity" with, for example, 177Lu-DOTATATE to select patients for targeted radionuclide therapy against these receptors (SSTR-PRRT)), a much higher proportion of patients were uPAR positive rather than SSTR positive, suggesting that more patients may be candidates for uPAR-targeted radionuclide therapy than those for SSTR-targeted radionuclide therapy. uPAR-positive lesions were identified in both low-grade (NET G1 / G2) and high-grade NEN (NET G3 and NEC) patients (Table 6). A much higher proportion of uPAR positivity was observed, especially in high-grade tumors, than reported as SSTR positive.

[0149] [Table 6] Data represents number of positive cases / total number of cases, percentages in brackets. *)UPAR target-to-liver ratio was used to calculate lesion SUV max / Normal Liver SUV mean Lesions were defined as uPAR positive if ≧1. #) SSTR positivity was defined as any area within the tumor with higher uptake than the liver (Yu J et al., Neuroendocrinology 2022).

[0150] conclusion uPAR PET is promising for the majority of patients, including those with high-grade disease, suggesting that most patients would be candidates for uPAR-targeted radionuclide therapy. This is in contrast to patients with SSTR-positive disease, whose tumors, especially high-grade tumors, are less promising.

[0151] Data Consideration UPAR PET ( 68 The main finding of this study on Ga-NOTA-AE105 (exemplified by uPAR) was that uPAR expression was observed in a large number of patients with low-grade and high-grade NENs. Moreover, high uPAR expression correlates with their poor prognosis in terms of both PFS and OS. These findings suggest that uPAR may be an attractive therapeutic target, both because of the availability of the target to patients with NENs and because of the possibility of specifically targeting lesions associated with poor patient outcomes.

[0152] The role of uPA and uPAR in cancer has been extensively studied in the past decades, and it is well established that higher expression of uPAR is associated with tumor growth, invasiveness, and metastatic spread, but its application in patients with NEN has not been fully studied (18). Therefore, several therapeutic approaches targeting uPA and uPAR have been investigated, including, for example, a uPAR antibody (huATN-658) (19) and a serine protease inhibitor targeting uPA (upamostat) (20). However, none of these therapeutic approaches have yet been approved for clinical use.

[0153] The aggressiveness of NEN varies greatly among patients. The primary tumor site, the presence or absence of metastasis, and the WHO classification are important prognostic markers and are used to guide treatment selection (21). Further studies by the present inventors and other groups have 64 Low somatostatin receptor density as determined by Cu-DOTATATE PET and 18High glucose metabolism, as determined by PET with F-FDG, has been suggested to be a prognostic factor (2, 3, 16, 22). The concept of tailored therapy is used to determine eligibility for targeted therapy using specific tumor markers. This concept has been widely implemented in the treatment of NEN patients, and somatostatin receptor imaging is used as an adjunct. 177 It has been used to screen for eligibility for somatostatin receptor-targeted therapy using Lu-DOTATATE. 177In a randomized trial comparing patients treated with Lu-DOTATATE with those treated with a high-dose cold somatostatin analog, the first group had significantly fewer deaths, longer PFS, and an 18% response rate (23). One drawback of targeting somatostatin receptor-expressing tumors is that their poor differentiation and more aggressive tumors have lower expression of somatostatin receptors (24). In contrast, uPAR expression is especially found in lesions that demonstrate tumor proliferation, invasiveness, and metastatic potential. Previously, our group investigated uPAR expression using immunohistochemical staining of primary tumors or metastases from patients with NEN G3 (Ki67>20%) and showed that 16 / 21 (76%) patients expressed uPAR in stromal and / or tumor cells (25). However, to the inventors' knowledge, uPAR expression in patients with low-grade NEN has never been studied in situ in vivo, but only indirectly by measuring soluble uPAR (suPAR) in serum (26). Since suPAR is a truncated version of membrane-bound uPAR, it could potentially be measured as a circulating uPAR biomarker. This study reported that suPAR levels were elevated in NEN patients compared to healthy controls, and in both low- and high-grade disease patients. However, no association between suPAR levels and overall survival was found. In contrast to this study, visualizing uPAR expression at the tumor level using PET is a more direct approach, allowing identification of lesions with highest uPAR expression and the location of these lesions. It has also been previously suggested that the expression pattern of uPAR is heterogeneous, such that uPAR is highly expressed at the tumor margins, thus promoting tissue invasion and metastatic dissemination locally, making it a hallmark of cancer (27). In support of this, we found that high uPAR expression on PET correlated with poorer prognosis in terms of both PFS and OS. In agreement with previous observations in NEC patients and the suPAR data in NEN, we found that uPAR expression is present in both low- and high-grade NEN. Thus, uPAR-targeted therapy may be applicable to patients with NEN of all grades.Our observations regarding uPAR expression must be taken into account, as the patients included in this study mainly had small intestinal or pancreatic primary tumors with metastatic disease and had been previously treated.

[0154] A potential but innovative perspective is to combine uPAR PET imaging and uPAR-PRRT as a theranostic pair to evaluate eligibility for uPAR-targeted therapy using uPAR PET. Although we have previously demonstrated high efficacy of uPAR PRRT in preclinical models of human prostate and colon cancer (11, 12), further studies are required to evaluate the feasibility of uPAR PRRT in NENs. However, the first step towards uPAR-PRRT for NENs would be to obtain evidence of high and specific uptake of uPAR-targeted radioligand in NENs and its prognostic impact, as was done in this study.

[0155] Data Overview uPAR expression assessed by PET with 68Ga-NOTA-AE105 was found in the majority of patients with both low- and high-grade NENs, and high uPAR expression may be associated with a poorer prognosis for them in terms of both PFS and OS. Taken together, this suggests that uPAR is an important target for risk stratification of patients with NENs and for targeted therapy.

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Claims

1. the imaging agent comprises a uPAR binding peptide bound to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA, the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; The uPAR binding mutant is selected from the group consisting of the following mutants: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile); selected from the group consisting of: If the quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TTR) exceeds a threshold level, a poor prognosis of recurrence-free survival (RFS) and / or overall survival (OS) is suggested: and When the quantitative level of said SUVmax and / or SUVmean and / or said target-to-liver ratio (TTR) is lower than a threshold level, it is suggested that the prognosis of recurrence-free survival (RFS) and / or overall survival (OS) is good; A positron emitting imaging agent for use in prognostic diagnosis of recurrence-free survival (RFS) and / or overall survival (OS) in patients with neuroendocrine tumors (NEN) undergoing PET imaging of their cancer.

2. 3. The imaging agent for use according to claim 1 or 2, wherein the peptide is (D-Asp)-(β-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser).

3. The formula:

3. The imaging agent for use according to claim 1 or 2, which is represented by the formula:

4. administering the imaging agent at a dose of 10-500 MBq, followed by a PET scan 10 minutes to 24 hours after administration of the imaging agent, and quantifying SUVmax and / or SUVmean; Use of an imaging agent according to any one of claims 1 to 3.

5. 5. The use of an imaging agent according to any one of claims 1 to 4, wherein the target to liver ratio (TLR) threshold level is in the range of 1.1 to 4, such as in the range of 1.3 to 4, such as in the range of 2 to 4 or 2 to 3.

6. 6. The imaging agent for use according to any one of claims 1 to 5, wherein the target to liver ratio (TLR) threshold level is in the range of 1.1 to 2, such as in the range of 1.1 to 1.5, such as in the range of 1.2 to 1.

4.

7. The imaging agent comprises a uPAR binding peptide bound to the radionuclide 68Ga or 64Cu via the chelator NOTA or DOTA; the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; if the quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) exceeds a threshold level, it is suggested that a uPAR-binding drug, such as a uPAR-binding radiopharmaceutical, is effective against said NEN; if the quantitative level of SUVmax and / or SUVmean and / or target-to-liver ratio (TLR) is lower than a threshold level, it is suggested that the uPAR-binding drug, such as a uPAR-binding radiopharmaceutical, is not effective against said NEN; The uPAR binding mutant is the following mutant: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile); Selected from the group consisting of: A positron-emitting imaging agent for use in performing companion diagnostics for patients with neuroendocrine tumors (NEN) who have undergone PET imaging of said cancer.

8. the radiopharmaceutical comprises a radionuclide and a uPAR binding peptide; and the uPAR-binding peptide is (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR-binding variant thereof; and The uPAR binding mutant is the following mutant: (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-([β]-2-naphthyl-L-alanine)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(Ser), (D-Glu)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([β]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([β]-1-naphthyl-L-alanine)-(D-His), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[β]ethoxyethyl)glycine), (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and (Asp)-([β]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile); selected from the group consisting of: The subject has been diagnosed with a uPAR-expressing neuroendocrine tumor (NEN); and said uPAR expression is above a predetermined threshold level; The threshold level is determined using a positron emitting imaging agent as defined in any one of claims 1 to 7; A composition comprising said radiopharmaceutical for use in treating or alleviating a neuroendocrine tumor (NEN) in said subject.

9. The radionuclide is for targeted radionuclide therapy and is selected from the group of isotopes consisting of 67Cu, 177Lu, 89Sr, 90Y, 117mSn, 131I, 153Sm, 166Ho, 186Re, 188Re, 211At, 212Pb, 212Bi, 213Bi, 223Ra, 224Ra, 225Ac, and 227Th; preferably selected from 177Lu, 67Cu, 90Y, 211At, 225Ac, and 227Th, more preferably 177Lu; A composition for use according to claim 8.

10. The radiopharmaceutical is attached to the uPAR binding peptide via a chelator, e.g., the chelator is selected from the group consisting of DOTA, CB-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A, TETA, TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, SarAr, SarAr-NCS, diamSar, AmBaSar, BaBaSar, ATSM, CB-TE1A1P and CB-TE2P, NOTA, NETA, TACN-TM, NODAGA, TRAP, AAZTA, DATA, H2dedpa, CP256, PCTA, THP, DTPA, 1B4M-DTPA, CHX-A ” -DTPA, TRAP (PRP9), NOPO, DFO HOPO, selected from the group consisting of H6phospa, PCTA, H2dedpa, H4octapa, H2azapa, H5decapa, HBED, HBED-cc, SHBED, BPCA, CP256, HEHA, PEPA and RESCA1; preferably selected from DOTA, NOTA, CB-TE2A, NODAGA, DFO, HBED and HBED-cc; more preferably said chelator is DOTA or NOTA; A composition for use according to claim 8 or 9.

11. A composition for use according to any one of claims 8 to 10, wherein said radiopharmaceutical is 177Lu-DOTA-AE105.

12. 12. The composition for use according to any one of claims 8 to 11, wherein the threshold level of the target to liver ratio (TLR) is in the range of 1.1 to 4, such as in the range of 1.3 to 4, for example in the range of 2 to 4 or 2 to 3, or wherein the threshold level of the target to liver ratio (TLR) is in the range of 1.1 to 2, such as in the range of 1.1 to 1.5, for example in the range of 1.2 to 1.4.

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