WO2025003190A1

WO2025003190A1 - Urokinase-type plasminogen activator receptor (upar)-pet/ct in brain tumours

Info

Publication number: WO2025003190A1
Application number: PCT/EP2024/067906
Authority: WO
Inventors: Andreas Kjaer
Original assignee: Curasight AS
Current assignee: Curasight AS
Priority date: 2023-06-27
Filing date: 2024-06-26
Publication date: 2025-01-02
Anticipated expiration: 2025-12-27

Abstract

The present invention relates to positron-emitting imaging agents for use in the prognosis of brain tumours in a patient by PET imaging of the cancer, wherein said imaging agent comprises an uPAR binding peptide coupled to a radionuclide. The invention also relates to compositions comprising a radiopharmaceutical for use in the treatment or alleviation of a brain tumour in a subject, wherein said radiopharmaceutical comprises a radionuclide and an uPAR binding peptide.

Description

UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (UPARJ-PET/CT IN BRAIN TUMOURS

Technical field of the invention

The present invention relates to a positron-emitting imaging agent for use in the prognostication of a brain cancer patient by PET imaging of the cancer, wherein said imaging agent comprises a uPAR binding peptide coupled via the chelating agent NOTA or DOTA to the radionuclide ⁶⁸Ga or ⁶⁴Cu. The invention also relates to compositions comprising a radiopharmaceutical for use in the treatment or alleviation of a brain cancer in a subject, wherein said radiopharmaceutical comprises a radionuclide and a uPAR binding peptide. Finally, the invention relates to the use of said imaging agents as companion diagnostic for the radiopharmaceutical for therapy.

Background of the invention

Gliomas are among the most common types of brain cancers, with an annual incidence of 6 cases per 100.000 individuals (1). These highly heterogenous tumors are graded in a multilayered approach into 4 distinct WHO grades. Grade 1-2 gliomas are referred to as low-grade (LGG) while grade 3-4 tumors are referred to as high-grade gliomas (HGG). Increasing WHO grade is correlated with increased tumor aggressiveness and poorer survival (2-4). In the era of many oncological advances, survival among patients with gliomas remains essentially unchanged with a 5-year survival rate of 82% for LGG to the most dismissal survival expectancy of 3-10 % among HGG (4-6). The treatment of gliomas is highly variable depending on tumor subtype. For LGGs treatment varies from watchful waiting after surgery (biopsy, partial, or gross total resection) to radiotherapy alone or concomitant chemotherapy including a procarbazine, lomustine, vincristine regime (PCV) or temozolomide (TMZ). For HGG, treatment aims at gross total tumor resection followed by concomitant radiotherapy and chemotherapy with TMZ or PCV (3). This variability in treatment regimens underlines the need for phenotyping and risk stratification of gliomas before treatment initiation in order to ensure more precise management of these tumors.

Magnetic resonance imaging (MRI) is the standard imaging modality to detect gliomas and can be complemented by positron emission tomography (PET), where particularly the use of amino acid tracers, such as O-(2-[¹⁸F]fluoroethyl)-l-tyrosine (FET), has been recommended (7-9). FET-PET has multiple applications, including diagnosis, prognostication, target delineation, and determination of tumor recurrence (9).

Additionally, PET imaging with the tracer [⁶⁸Ga]Ga-NOTA-Asp-Cha-Phe-D-Ser-D- Arg-Tyr-Leu-Trp-Ser-OH (⁶⁸Ga-NOTA-AE105) targeting the proteolytic urokinase plasminogen activator (uPA) system is emerging as a promising new imaging biomarker for diagnosis, prognostication, risk stratification as well as a therapeutic target for solid cancers. Over the years, several studies have shown the applicability of uPA receptor (uPAR) as a diagnostic biomarker in cancer associated with poor disease prognosis (10). uPAR is highly upregulated in most solid cancers with limited expression in normal tissue. It is located on the surface of the cell where it binds the serine protease uPA. This facilitates cell proliferation, angiogenesis, proteolysis, motility resulting in tumor progression and invasion into the surrounding tissue 10-12).

To target uPAR, the PET radiotracers ⁶⁸Ga-NOTA-AE105 and ⁶⁴Cu-DOTA-AE105 where the targeting peptide is a high-affinity antagonist for uPAR was developed (13-15). The safety, biodistribution, and radioligand accumulation of ⁶⁸Ga-NOTA- AE105 and ⁶⁴Cu-DOTA-AE105 in cancer tissue in two Phase 1 trials involving primary tumors and metastases has previously been established. Tracer accumulation was histopathologically confirmed to correspond with cancer tissue and uPAR expression using immunohistochemistry (13, 36). Furthermore, the utility of ⁶⁸Ga-NOTA-AE105 for uPAR-PET as a promising method for non-invasive evaluation of localized prostate cancer with high diagnostic accuracy in differentiating between low-risk and intermediate-risk Gleason score profiles have been demonstrated (16). uPAR-PET has also been found to be highly prognostic in neuroendocrine neoplasms (17) and head-and-neck cancer (18). In gliomas, uPAR-PET has been highlighted as an effective imaging biomarker for tumour visualization using an orthotopic human xenograft model of glioblastoma (19).

From a therapeutic perspective, uPAR has been identified as a promising target for peptide receptor radionuclide therapy (PRRT) and therapeutic efficacy of uPAR- targeted PRRT in preclinical models of prostate and colorectal cancers has previously been demonstrated the (20,21). Moreover, recent work has revealed a high correlation between uPAR expression on uPAR-PET and both Overall Survival (OS) and Progression-Free Survival (PFS) in patients with neuroendocrine neoplasms underscoring uPAR as a promising target for PRRT treatment. Actually, 68% of these patients across tumor grades were uPAR positive (17).

Hence, improved prognostic tools in relation to brain tumours would be advantageous, and in particular a more efficient and/or reliable treatments of subtypes of brain tumours would be advantageous.

Summary of the invention

It has been found that uPAR-PET can serve as a prognostic marker of tumor aggressiveness in brain cancers, such as gliomas, and that uPAR-PET positive brain cancers may be future candidates for uPAR-targeted peptide receptor radionuclide therapy (PRRT).

Thus, an aim of this study with uPAR-targeted PRRT (exemplified with ⁶⁸Ga-NOTA- AE105) PET/MRI in patients with primary gliomas was to investigate the association between the uptake of uPAR-targeted PRRT on both OS and PFS. Furthermore, an aim was to determine the proportion of uPAR-PET positive tumours to assess how many of these patients could potentially be eligible for future uPAR-PRRT.

Thus, an object of the present invention relates to the provision of improved prognostic tools for brain tumors.

In particular, it is also an object of the present invention to provide improved treatment protocols for patients suffering from brain tumors.

Thus, one aspect of the invention relates to a positron-emitting imaging agent for use in the prognostication of Progression-Free Survival (PFS) and/or Overall Survival (OS) of a brain tumour in a patient by PET imaging of the cancer, wherein said imaging agent comprises a uPAR binding peptide coupled to a radionuclide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; wherein the uPAR binding variant is selected from the group consisting of

• (D-Asp)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)-(Trp)-(Ser),

• (Ser)-(Leu)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(Gln)- (Tyr)(Leu)-(Trp)-(Ser),

• (D-Glu)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(D-Tyr)- (Tyr)-(Leu)-(Trp)-(Ser),

• (Asp)-( [beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)-(Trp)-(Ser),

• (Asp)-( [beta] -cyclohexyl-L-a Ian ine)-(Phe)-(Ser)-(D-Arg)-(Tyr)- Leu)-(Trp)-(Ser),

• (D-Asp)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(Ser)-(D-Arg)- (Tyr)-Leu)-(Trp)-(Ser),

• (D-Th r)-([ beta] -cyclohexyl-L-a la n ine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)-(Trp)-(Ser),

• (D-Asp)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)-([ beta] -2-naphthyl-L-a Ian ine)-(Ser),

• (Asp)-( [beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(Arg)-(Tyr)- (Leu)-(Trp)-(Ser),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)([beta]-l-naphthyl-L-alanine)-(Ser),

• (D-Glu)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(Tyr)- (Tyr)-(Leu)-(Trp)-(Ser),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- (Leu)-(Leu)-(Trp)-(D-His),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- ([beta]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile), • (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)([beta]-l-naphthyl-L-alanine)-(D-His),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3- indolylethyl)glycine)-(N-(2-methoxyethyl)glycine),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N- (2 [beta]thoxyethyl)g lycine),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N- (methylnaphthalyl)glycine)-(N-(2-methoxyethyl)glycine), and

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3- dimethoxybenzyl)glycine)-(Ile); wherein a SUVmax and/or SUVmean (quantification) level above a threshold level is indicative of a poor Progression-Free Survival (PFS) prognosis and/or poor Overall Survival (OS) prognosis; and wherein a SUVmax and/or SUVmean (quantification) level equal to or below a threshold level is indicative of good Progression-Free Survival (PFS) prognosis and/or good Overall Survival (OS) prognosis.

The two uPAR-PET tracers ⁶⁴Cu-DOTA-AE105 and 68Ga-NOTA-AE105 share the same binding moiety, the peptide AE105. In addition, they have both been tested in both humans and animals (see e.g. WO2014/086364 Al) and have demonstrated similar uptake in breast, bladder and prostate cancer. Accordingly, without being bound by theory, it is believed PET tracers comprising

- ⁶⁴Cu or ⁶⁸Ga as radionuclide;

- DOTA or NOTA as chelator; and

- the uPAR binding peptide according to the present invention; will work in a similar manner also for brain tumor patients. Preferably said imaging agent comprises a uPAR binding peptide coupled via the chelating agent NOTA to the radionuclide 68Ga.

As outlined above, the methods of the invention and the compositions for use according to the invention, may also find use as a companion diagnostic.

Thus, a further aspect of the invention relates to a positron-emitting imaging agent for use as companion diagnostic of a brain tumour patient by PET imaging of the cancer, wherein said imaging agent comprises a uPAR binding peptide coupled to a radionuclide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; wherein o a SUVmax and/or SUVmean level above a threshold level is indicative of that a uPAR binding drug, such as a uPAR binding radiopharmaceutical will be effective against said brain tumour; and o a SUVmax and/or SUVmean level equal to or below a threshold level is indicative of that a uPAR drug, such as a uPAR binding radiopharmaceutical will not be effective against said brain tumour; wherein the uPAR binding variant is selected from the group consisting of

• (Asp)-( [beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)-(Trp)-(Ser), • (Asp)-( [beta] -cyclohexyl-L-a Ian ine)-(Phe)-(Ser)-(D-Arg)-(Tyr)- Leu)-(Trp)-(Ser),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- ([beta]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)([beta]-l-naphthyl-L-alanine)-(D-His),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3- dimethoxybenzyl)glycine)-(Ile). Yet a further aspect of the invention relates to a composition comprising a radiopharmaceutical for use in the treatment or alleviation of a brain tumor in a subject; wherein said radiopharmaceutical comprises a radionuclide and a uPAR binding peptide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; and wherein the uPAR binding variant is selected from the group consisting of

• (D-Glu)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(Tyr)- (Tyr)-(Leu)-(Trp)-(Ser), • (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- (Leu)-(Leu)-(Trp)-(D-His),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3- dimethoxybenzyl)glycine)-(Ile); wherein said subject has been diagnosed with a uPAR expressing brain tumor and wherein said uPAR expression is above a predetermined threshold level, wherein said threshold level is determined using a positron-emitting imaging agent as defined according to this invention.

Brief description of the figures

Figure 1

Figure 1 shows consolidated Standards of Reporting Trials (CONSORT) flow diagram of inclusion process.

Figure 2

Figure 2 shows examples of uPAR PET/MRI performed on patient with glioblastoma, IDH-wildtype, WHO Grade 4 (MGMT non-methylated) in the temporoparietal lobe with tumor SUVmax 3.3. A) T1W MPRAGE MRI with gadolinium contrast. B) T2W FLAIR MRI image. C) uPAR-PET image. D) Merged T2W FLAIR MRI and uPAR-PET image. Color scale from 0 to tumor SUVmax value of 3.3.

Figure 3

Figure 3 shows Examples of uPAR PET/MRI performed on patient with glioblastoma, IDH-wildtype, WHO Grade 4 (MGMT non-methylated) involving the genu corpus callosum with tumor SUVmax 2.2. A) T1W MPRAGE MRI with gadolinium contrast. B) T2W FLAIR MRI image. C) UPAR-PET image. D) Merged T2W FLAIR MRI and uPAR-PET image. Color scale from 0 to tumor SUVmax value of 2.2.

Figure 4

Figure 4 shows primary gliomas Kaplan Meier Survival plots of (A) Overall Survival (OS) dichotomized at SUVmax 1.1 and (B) Progression-Free Survival (PFS) dichotomized at SUVmax 0.635.

Figure 5

Figure 5 shows primary HGG Kaplan Meier Survival plots of (A) Overall Survival (OS) and (B) Progression-Free Survival (PFS) both dichotomized at SUVmax 1.1.

The present invention will now be described in more detail in the following.

Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Brain tumors

A brain tumour occurs when abnormal cells form within the brain. Gliomas are among the most common types of brain cancers. Gliomas can be divided into high-grade gliomas (HGG) (WHO grade 3 and 4) and low-grade gliomas (LGG) (WHO grade 1+2). WHO grade 4 are also named glioblastoma and constitute the main part (around 80%) of high-grade gliomas. ⁶⁸Ga

Gallium-68.

⁶⁴Cu

Copper-64.

¹⁷⁷ Lu

¹⁷⁷Lu is a low-energy beta-emitter (~1.5 mm maximum penetration in soft tissue) capable of inducing cytotoxic effects in tumors but not or very limited in surrounding tissue by providing both a 'cross-fire'- and 'bystander' effect from direct betaparticles and Auger electrons, respectively. ¹⁷⁷Lu therefore is considered an optimal radionuclide for therapy of large as well as small tumor lesions and/or disseminated metastatic disease.

AE105

Ac-Asp-Cha-Phe-(D)Ser-(D)Arg-Tyr-Leu-Trp-Ser

The peptide according to the invention can e.g. be synthesized by standard solidphase peptide chemistry.

NOTA

NOTA: 2,2',2"-(l,4,7-triazacyclononane-l,4,7-triyl)triacetic acid.

NOTA may be coupled to AE105 thereby providing NOTA-AE105 (NOTA-Asp-Cha- Phe-Ser-Arg-Tyr-Leu-Trp-Ser)). This can be illustrated by the following chemical structure:

In an embodiment, the imaging agent is 68Ga-NOTA-AE105.

DOTA

DOTA (also known as tetraxetan) is an organic compound with the formula (CH₂CH₂NCH₂CO₂H)₄. 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. Its complexes have medical applications as contrast agents and cancer treatments.

Preferred IUPAC name of DOTA 2,2',2",2"'-(l,4,7,10-Tetraazacyclododecane-l,4,7,10-tetrayl)tetraacetic acid Further below DOTA is shown in complex with ⁶⁸Ga and ⁶⁴CU coupled to AE105.

Progression-Free Survival CPFS)

The term "progression-free survival" (PFS) is defined as "the length of time during and after the treatment or intervention of a disease, such as cancer, that a patient lives with the disease but it does not get worse or progresses".

The term "relapse-free survival" (RFS) is defined as clinical endpoint defined as time from diagnosis to any relapse of the disease at the locoregional (TN site) and/or distant metastasis (M site) with deaths from other causes recorded as censoring and disease-free survival (DFS), which is defined as RFS, but includes death of any reason as an event. Overall Survival ( OS)

The term "Overall Survival" (OS) is defined as time from diagnosis to death of any cause.

Threshold level

In the context of the present invention, the term "threshold level", "reference level" or "cut-off" relates to a standard in relation to a quantity, which other values or characteristics can be compared to.

In one embodiment of the present invention, it is possible to determine a threshold level by investigating the uPAR levels by PET/CT from healthy subjects. By applying different statistical means, such as cut-off finding, multivariate analysis, one or more threshold level can be calculated.

See also example 5, where cut-offs are determined.

Based on these results, a cut-off may be obtained that shows the relationship between the level(s) detected and patients at risk. The cut-off can thereby be used e.g. to determine the uPAR levels, which for instance corresponds to an increased risk of a poor PFS or OS.

Risk Assessment

The present inventors have successfully developed a new method to predict the prognosis of a subject with brain cancer, such as PFS and/or RFS and/or OS. To determine whether a patient has an increased risk of a poor prognosis, a cut-off (reference level) must be established. This cut-off may be established by the laboratory, the physician or on a case-by-case basis for each patient.

The cut-off level could be established using a number of methods, including: multivariate statistical tests (such as partial least squares discriminant analysis (PLS-DA), random forest, support vector machine, etc.), percentiles, mean plus or minus standard deviation(s); median value; fold changes.

The multivariate discriminant analysis and other risk assessments can be performed on the 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.

As obvious to one skilled in the art, in any of the embodiments discussed above, changing the risk cut-off level could change the results of the discriminant analysis for each subject.

Statistics enables evaluation of the significance of each level. Commonly used statistical tests applied to a data set include t-test, f-test or even more advanced tests and methods of comparing data. Using such a test or method enables the determination of whether two or more samples are significantly different or not. The significance may be determined by the standard statistical methodology known by the person skilled in the art.

The chosen reference level may be changed depending on the mammal/subject for which the test is applied.

Preferably, the subject according to the invention is a human subject.

The chosen reference level may be changed if desired to give a different specificity or sensitivity as known in the art. Sensitivity and specificity are widely used statistics to describe and quantify how good and reliable a biomarker or a diagnostic test is. Sensitivity evaluates how good a biomarker or a diagnostic test is at detecting a disease, while specificity estimates how likely an individual (i.e. control, patient without disease) can be correctly identified as not at risk.

Several terms are used along with the description of sensitivity and specificity; true positives (TP), true negatives (TN), false negatives (FN) and false positives (FP). If a disease is proven to be present in a sick patient, the result of the diagnostic test is considered to be TP. If a disease is not present in an individual (i.e. control, patient without disease), and the diagnostic test confirms the absence of disease, the test result is TN. If the diagnostic test indicates the presence of disease in an individual with no such disease, the test result is FP. Finally, if the diagnostic test indicates no presence of disease in a patient with disease, the test result is FN.

As used herein the sensitivity refers to the measures of the proportion of actual positives, which are correctly identified as such, i.e. the percentage of subjects having a risk of a poor prognosis above normal who are identified as having a poor prognosis above normal.

Usually the sensitivity of a test can be described as the proportion of true positives of the total number with the target disorder i.e. a risk of poor prognosis above normal. All patients with the target disorder are the sum of (detected) true positives (TP) and (undetected) false negatives (FN).

As used herein the specificity refers to measures of the proportion of negatives which are correctly identified - i.e. the percentage of mammal with a nonincreased risk of poor prognosis that are identified as not having a risk of poor prognosis above normal. The ideal diagnostic test is a test that has 100 % specificity, i.e. only detects subjects with a risk of having poor prognosis above normal and therefore no false positive results, and 100% sensitivity, i.e. detects all subjects with a risk of having poor prognosis above normal and therefore no false negative results.

For any test, there is usually a trade-off between each measure. For example, in a manufacturing setting in which one is testing for faults, one may be willing to risk discarding functioning components (low specificity), in order to increase the chance of identifying nearly all faulty components (high sensitivity). This trade-off can be represented graphically using a ROC curve.

The chosen specificity determines the percentage of false positive cases that can be accepted in a given study/population and by a given institution. By decreasing specificity an increase in sensitivity is achieved.

As will be generally understood by those skilled in the art, methods for screening for prognosis are processes of decision making and therefore the chosen specificity and sensitivity depends on what is considered to be the optimal outcome by a given institution/clinical personnel. SUVmax

In the present context, the term "SUVmax" refers to the "maximum standardized uptake value" (SUVmax), which is widely used for measuring the uptake of uPAR and FDG by malignant tissue. Increased uptake values reflect increased uptake or binding of the tracers to cancer cells, and can be imaged and quantified using PET.

SUVmean

In the present context the term "SUVmean", refers to the mean standardized uptake value.

Companion diagnostic

In the present context, a "companion diagnostic" is a diagnostic test used as a companion to a therapeutic drug to determine its applicability to a specific person.

Companion diagnostics may be co-developed with drugs to aid in selecting or excluding patient groups for treatment with that particular drug based on their biological characteristics that determine responders and non-responders to the therapy. Companion diagnostics are developed based on companion biomarkers, biomarkers that prospectively help predict likely response or severe toxicity.

Imaging agent for use in the prognostication of a brain cancer patient by PET

Improved prognostication of brain cancer patients are important, e.g. for determining the need for radiotherapy, but also for determining the correct radiotherapy. Thus, improved companion diagnostics are also important. Thus, one aspect of the invention relates to a positron-emitting imaging agent for use in the prognostication of Progression-Free Survival (PFS) and/or Overall Survival (OS) of a brain tumour in a patient by PET imaging of the cancer, wherein said imaging agent comprises a uPAR binding peptide coupled to a radionuclide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; wherein the uPAR binding variant is selected from the group consisting of

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)([beta]-l-naphthyl-L-alanine)-(D-His), • (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(3- indolylethyl)glycine)-(N-(2-methoxyethyl)glycine),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3- dimethoxybenzyl)glycine)-(Ile); wherein a SUVmax and/or SUVmean level above a threshold level is indicative of a poor Progression-Free Survival (PFS) prognosis and/or poor Overall Survival (OS) prognosis; and wherein a SUVmax and/or SUVmean and/or level equal to or below a threshold level is indicative of good Progression-Free Survival (PFS) prognosis and/or good Overall Survival (OS) prognosis.

As outlined in example 5, the imaging agent according to the invention is a strong prognostic marker for brain cancer patients. Further, optimized cut-offs have been identified. Again, it is noted that such marker is also relevant as a companion diagnostics, since it can be used for identifying patients, which are likely susceptible to radiotherapy using a radionuclide coupled to a uPAR binding peptide.

Different uPAR binding variants of the uPAR binding peptide (including AE105) are disclosed in WO2014/086364 Al.

The uPAR binding part of the imaging agent is preferably the one known as AE105. Thus, in an embodiment, the peptide is (D-Asp)-([beta]-cyclohexyl-L- alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser). In an embodiment, the radionuclide is selected from the group consisting of ⁶⁸Ga and ⁶⁴Cu.

In another embodiment, the radionuclide is coupled to the uPAR binding peptide via a chelating agent, such as DOTA or NOTA.

In a preferred embodiment, the radionuclide is ⁶⁸Ga coupled to the uPAR binding peptide via a chelating agent NOTA. In another preferred embodiment, the imaging agent for use has the formula

In yet another embodiment, the radionuclide is ⁶⁴Cu coupled to the uPAR binding peptide via a chelating agent DOTA or NOTA.

In a preferred embodiment, the imaging agent for use has the formula

In another preferred embodiment, the imaging agent for use has the formula

In an embodiment, the imaging agent is to be administered in a dose of 10- 500 MBq followed by PET scanning 10 min to 24 hours after the imaging agent has been administered, and quantification through SUVmax and/or SUVmean. A brain tumour occurs when abnormal cells form within the brain. Gliomas are among the most common types of brain cancers. Gliomas can be divided into high-grade gliomas (HGG) (WHO grade 3 and 4) and low-grade gliomas (LGG) (WHO grade 1+2). WHO grade 4 are also named glioblastoma and constitute the main part (around 80%) of high-grade gliomas. In an embodiment, the brain tumor is a high-grade glioma (WHO grade 3 and 4) or a low-grade glioma (WHO grade 1 and 2), preferably a high-grade glioma WHO grade 4 (also known as glioblastoma). As shown in example 3, using the method of the invention, WHO grade 4 are efficiently imaged. In addition, most of the cancers imaged were WHO grade 4.

In another embodiment, the C-terminal is a carboxylic acid or an amide.

To further improve localization of the cancer, the PET scan may be combined with other scanning types. Thus, in an embodiment, prognostication comprises PET/CT scanning and/or PET/MR scanning. In the example section, PET/CT has been used.

The prognostication may be further defined. Thus, in an embodiment, the prognosis is prognosis of Progression-Free Survival (PFS) and/or Overall Survival (OS). In the example section, PFS and OS have been assessed.

Different amount (MBq) of the imaging may be used. Also the time before scanning after administration of the imaging agent may vary. Thus, in an embodiment, the imaging agent is to be administered in a dose of 10-500 MBq followed by PET scanning 10 min to 24 hours after the imaging agent has been administered, and quantification through SUVmax and/or SUVmean and/or TLR.

In another embodiment, the imaging agent is to be administered in a dose of 20-400 MBq, such as 50-400 MBq, such as 70-300 MBq, such as 100-300 MBq or such as 100-300 MBq. preferably such as 150-250 MBq, and more preferably 170-230 MBq. Thus in a preferred embodiment, the imaging agent is to be administered in a dose of 70-300 MBq, preferably such as 150-250 MBq. In the example section, around 200 MBq has been used and PET imaging was performed with a Siemens Biograph 128 mCT. However, other equipment may be more sensitive and therefore allow for lower amount (MBq) of the imaging agent In an embodiment, a sufficient amount of imaging aging is administered to allow for PET imaging with enough radioactivity dose for imaging.

⁶⁸Ga has a half-life of around 1 hour (68 min) making 10 minutes to 5 hours, preferably 20 minutes to 3 hours realistic time before PET scanning. ⁶⁴Cu has a half-life of around 12.7 hours making an interval of 20 minutes to 24 hours before PET scanning realistic.

Thus, in a related embodiment, the PET scanning follows 20 minutes to 10 hours after the imaging agent has been administered, such as 20 minutes to 5 hours, such as 30 minutes to 3 hours after the imaging agent has been administered.

In yet another embodiment, the imaging agent according to the invention is in a pharmaceutical composition comprising the imaging agent, together with one or more pharmaceutical acceptable adjuvants, excipients and/or diluents.

To be able to make a prognostication, a threshold level (cut-off/reference level) may be included. Thus, in an embodiment, the imaging agent for use according to the invention, o a SUVmax and/or SUVmean level above a threshold level is indicative of a poor Progression-Free Survival (PFS) prognosis and/or poor Overall Survival (OS) prognosis; and o a SUVmax and/or SUVmean level equal to or below a threshold level is indicative of good Progression-Free Survival (PFS) prognosis and/or good Overall Survival (OS) prognosis.

As also outlined in the example section, "cut-offs" /"threshold levels", were identified using the Cutoff Finder application (26).

In example 5, specific optimal threshold levels (cut-offs) have been calculated for ⁶⁸Ga-NOTA-AE105. Thus, in another embodiment, said SUVmax and/or SUVmean threshold level is in the range 0.5-4, such as in the range 0.5-2, preferably in the range 0.5-1.5. In example 5, optimal threshold levels for both PFS and OS were determined.

In an embodiment, said SUVmax and/or SUVmean threshold in relation to PFS for all gliomas is in the range 0.3-1, preferably 0.4-0.8, more preferably around 0.64.

In an embodiment, said SUVmax and/or SUVmean threshold in relation to PFS for high-grade gliomas is in the range 0.7-1.5, preferably 0.9-1.3, more preferably around 1.1.

In an embodiment, said SUVmax and/or SUVmean threshold in relation to OS for all gliomas is in the range 0.7-1.5, preferably 0.9-1.3, more preferably around 1.1.

In an embodiment, said SUVmax and/or SUVmean threshold in relation to OS for all high-grade gliomas is in the range 0.7-1.5, preferably 0.9-1.3, more preferably around 1.1.

In a preferred embodiment, the above values are for ⁶⁸Ga-NOTA-AE105

For ⁶⁴Cu-DOTA-AE105 2-4-fold higher SUVmax and or SUVmean values are seen and the stated cut-off values will likewise be a factor 2-4 higher than stated above.

Thus in another embodiment, said SUVmax and/or SUVmean threshold level is in the range 2-16, such as in the range 2-8, preferably in the range 2-6, preferably when ⁶⁴Cu-DOTA-AE105 is used.

In an embodiment, said SUVmax and/or SUVmean threshold in relation to PFS for all gliomas is in the range 0.6-4, preferably 0.8-3.2, more preferably around 1.2- 2.4, preferably when ⁶⁴Cu-DOTA-AE105 is used.

In an embodiment, said SUVmax and/or SUVmean threshold in relation to PFS for high-grade gliomas is in the range 2.8-24, preferably 3.6-20.8, more preferably around 4.4-17.6, preferably when ⁶⁴Cu-DOTA-AE105 is used.. In an embodiment, said SUVmax and/or SUVmean threshold in relation to OS for all gliomas is in the range 1.4-6, preferably 1.8-5.2, more preferably around 2.2- 4.4, preferably when ⁶⁴Cu-DOTA-AE105 is used.

In an embodiment, said SUVmax and/or SUVmean threshold in relation to OS for all high-grade gliomas is in the range 1.4-6, preferably 1.8-5.2, more preferably around 2.2-4.4, preferably when ⁶⁴Cu-DOTA-AE105 is used.

In yet an embodiment, said threshold level is determined by using a cut-off finding method to obtain a split in a Kaplan-Meier plot (log-rank test) and the corresponding hazard ratios (HRs).

It is of course noted that embodiments of other aspects of the invention is also applicable to this aspect.

A method of in vivo imaging by PET imaging, for assessing the prognosis of a brain cancer

Another aspect of the invention relates to a method of in vivo imaging by PET imaging, for assessing the prognosis of a brain cancer in a patient, said method comprising: a) provision of a subject to which a imaging agent according to the invention has been previously administered; b) detecting by in vivo PET imaging the radioactive emissions from the radioisotope of the administered imaging agent of step a); 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, wherein said expression is correlated with said signals emitted by said in vivo imaging agent; and e) comparing the determined distribution and extent of uPAR expression to a threshold level;

■ wherein a SUVmax and/or SUVmean level above a threshold level is indicative of a poor Progression-Free Survival (PFS) prognosis and/or poor Overall Survival (OS) prognosis; and wherein a SUVmax and/or SUVmean level equal to or below a threshold level is indicative of good Progression-Free Survival (PFS) prognosis and/or good Overall Survival (OS) prognosis.

In an embodiment, if a good Progression-Free Survival (PFS) prognosis or good Overall Survival (OS) prognosis is indicated, said subject will be scheduled for a less aggressive treatment regimen to avoid unnecessary toxicity. The skilled person in the field will be well aware of different less aggressive treatment regimes compared to the treatment already in place for the patient in questions. Examples are changed treatment compounds (or radiation), lower dosages or interval between treatments.

In a further embodiment, a level expressed as SUVmax, SUVmean determines if a patient is eligible for targeted radionuclide therapy.

In an aspect, the method forms the basis as a companion diagnostics for targeted radionuclide therapy. Thus, in such as an aspect o a SUVmax and/or SUVmean level above a threshold level is indicative of that a uPAR binding drug, such as a uPAR binding radiopharmaceutical will be effective against said brain cancer; and o a SUVmax and/or SUVmean level equal to or below a threshold level is indicative of that a uPAR binding drug, such as a uPAR binding radiopharmaceutical will not be effective against said brain cancer.

Preferably, the drug is a uPAR binding radiopharmaceutical, more preferably the radiopharmaceutical is ¹⁷⁷Lu-DOTA-AE105, ⁶⁷Cu-DOTA-AE105, ²²⁵Ac- DOTA-AE105, or ²¹²Pb-DOTA-AE105.

Use of a positron-emitting imaging agent in the prognosis of a brain cancer in a patient by in vivo PET imaging

A further aspect of the invention relates to the use of a positron-emitting imaging agent according to the invention in the prognosis of brain cancer patients by in vivo PET imaging of uPAR expressing tumors, wherein said imaging agent comprises a uPAR binding peptide coupled via the chelating agent NOTA to the radionuclide ⁶⁸Ga; wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof.

In an alternative part of this aspect, said imaging agent comprises a uPAR binding peptide coupled via the chelating agent NOTA or DOTA to the radionuclide ⁶⁸Ga or ⁶⁴Cu.

Again, in an embodiment, the uPAR binding variant thereof is selected from the group consisting of

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3- dimethoxybenzyl)glycine)-(Ile).

Companion diagnostic

As outlined above, the methods of the invention and the compositions for use according to the invention, may also find use as a companion diagnostic. Thus, a further aspect of the invention relates to a positron-emitting imaging agent for use as companion diagnostic of a brain tumour patient by PET imaging of the cancer, wherein said imaging agent comprises a uPAR binding peptide coupled to a radionuclide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; wherein o a SUVmax and/or SUVmean level above a threshold level is indicative of that a uPAR binding drug, such as a uPAR binding radiopharmaceutical will be effective against said brain tumour; and o a SUVmax and/or SUVmean level equal to or below a threshold level is indicative of that a uPAR drug, such as a uPAR binding radiopharmaceutical will not be effective against said brain tumour; wherein the uPAR binding variant is selected from the group consisting of

In a preferred embodiment, the imaging agent is ⁶⁸Ga-NOTA-AE105. Thus, in an embodiment, the radionuclide and chelator are ⁶⁸Ga-NOTA. This imaging agent has been used in the example section.

In another preferred embodiment, the imaging agent is ⁶⁴Cu-DOTA-AE105 Thus, in yet another embodiment, the radionuclide and chelator are ⁶⁴Cu- DOTA.

In a further embodiment, the positron-emitting imaging agent is a companion diagnostic for a radiopharmaceutical.

In a preferred embodiment, the positron-emitting imaging agent is a companion diagnostic for a radiopharmaceutical as defined according to the present invention.

In yet another preferred embodiment, the positron-emitting imaging agent is a companion diagnostic for a radiopharmaceutical as defined according to the present invention, wherein the radionuclide and chelator of the radiopharmaceutical is ¹⁷⁷Lu-DOTA. In a related preferred embodiment, the positron-emitting imaging agent is a companion diagnostic for a radiopharmaceutical having the formula :

As defined above, companion diagnostics can be used to determine the applicability of a drug. Thus, in an embodiment, the companion diagnostic is for determining the applicability of a radiopharmaceutical to a specific person, such as a radiopharmaceutical as defined according to the present invention.

In another preferred embodiment, the drug/ radiopharmaceutical is a radiopharmaceutical. In an even more preferred embodiment, the uPAR binding radiopharmaceutical is ¹⁷⁷Lu-DOTA-AE105, ⁶⁷Cu-DOTA-AE105, ²²⁵Ac- DOTA-AE105, and/or ²¹²Pb-DOTA-AE105.

In an embodiment, the brain tumor is a high-grade glioma (WHO grade 3 and

4) or a low-grade glioma (WHO grade 1 and 2), preferably a high-grade glioma (WHO grade 4) (glioblastoma). In yet an embodiment, the brain tumor is a high-grade glioma (glioblastoma).

Again, it is noted that embodiments of other aspects of the invention is also applicable to this aspect.

Radiopharmaceutical for use in the treatment or alleviation of a brain cancer in a subject

As outlined above, the imaging agent may be used as a companion diagnostic. In this way subtypes of brain cancers can be identified which expresses certain upregulated or downregulated surface proteins. Upregulated surface proteins may be used to target radiopharmaceutical to a cancer.

Thus, yet an aspect of the invention relates to a composition comprising a radiopharmaceutical for use in the treatment or alleviation of a brain tumor in a subject; wherein said radiopharmaceutical comprises a radionuclide and a uPAR binding peptide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; and wherein the uPAR binding variant is selected from the group consisting of

• (Asp)-( [beta] -cyclohexyl-L-a Ian ine)-(Phe)-(Ser)-(D-Arg)-(Tyr)- Leu)-(Trp)-(Ser), • (D-Asp)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(Ser)-(D-Arg)- (Tyr)-Leu)-(Trp)-(Ser),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3- dimethoxybenzyl)glycine)-(Ile); wherein said subject has been diagnosed with a uPAR expressing brain tumor and wherein said uPAR expression is above a predetermined threshold level, wherein said threshold level is determined using a positron-emitting imaging agent according to the invention.

Preferably, the radionuclide and the peptide are linked via a chelator, such as DOTA or NOTA.

In an embodiment, the radionuclide is selected from the group consisting of therapeutic alpha-emitters, therapeutic beta-emitters or therapeutic auger emitters.

In yet another embodiment, the radionuclide is for targeted radionuclide therapy (alpha, beta-emitters 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 selected from ¹⁷⁷Lu, ⁶⁷Cu, ⁹⁰Y, ²¹¹At, ²²⁵Ac, and ²²⁷Th, and more preferably being ¹⁷⁷Lu.

In another embodiment, said radiopharmaceutical is coupled to the uPAR binding peptide via a chelator, such as the chelator being 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, H6phospa, PCTA, H2dedpa, H4octapa, H2azapa, H5decapa, HBED, HBED-cc, SHBED, BPCA, CP256, HEHA, PEPA and RESCA1, preferably selected from any of DOTA, NOTA, CB-TE2A, NODAGA, DFO, HBED, and HBED-cc, more preferably the chelator is DOTA or NOTA.

In an embodiment, the radiopharmaceutical is selected from the group consisting of ¹⁷⁷Lu-DOTA-AE105, ⁶⁷Cu-DOTA-AE105, ²²⁵Ac-DOTA-AE105, and ²¹²Pb-DOTA-AE105.

In an embodiment, the radiopharmaceutical (radionuclide and chelator is selected from the group consisting of ¹⁷⁷Lu-DOTA, ⁶⁷Cu-DOTA, ²²⁵Ac-DOTA, and ²¹²Pb-DOTA. In a preferred embodiment, the radiopharmaceutical is ¹⁷⁷Lu-DOTA-AE105.

In another preferred embodiment, the radiopharmaceutical is ⁶⁷Cu-DOTA-AE105. In yet another embodiment, the radiopharmaceutical has the formula :

As also outlined in the discussion of the data below, the findings disclosed in here, highlight the potential of uPAR as a therapeutic target in gliomas and most importantly as a target for uPAR-PRRT. In particular, the positive uptake on uPAR- PET suggests that uPAR-PRRT labelled with a therapeutic alpha or beta emitter, may be administered systemically rather than intratumorally. Thus, in an embodiment, the radiopharmaceutical is administered systemically or intratumorally.

In yet an embodiment, the radiopharmaceutical is administered systemically. In an embodiment, the brain tumor is a high-grade glioma (WHO grade 3 and 4) or a low-grade glioma (WHO grade 1 and 2), preferably a high-grade glioma (WHO grade 4) (glioblastoma).

In yet an embodiment, the brain tumor is a high-grade glioma (glioblastoma).

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1 - Materials and methods

Study Design

In this prospective clinical trial, eligible patients were enrolled from the Department of Neurosurgery at Copenhagen University Hospital, Rigshospitalet, between March 2017 and June 2022. Patients were eligible if they met the following inclusion criteria; more than 18 years of age, able to read and understand the patient information in Danish and give informed consent; had a newly diagnosed intracranial lesion suspected of primary glioma on brain MRI; and were scheduled for neurosurgery (biopsy or tumor resection).

Patients were excluded if they were pregnant or breast-feeding, had a body weight above 140 kg, had claustrophobia, age above 85 years, or suspected of allergy to ⁶⁸Ga-NOTA-AE105.

Written informed consent was obtained from all patients. Following consent, the patients were scheduled for a ⁶⁸Ga-NOTA-AE105 PET/MRI brain scan before neurosurgery.

PET/MRI Acquisition The tracer ⁶⁸Ga-NOTA-AE105 was synthesized as previously described 13). PET/MRI scan with the radiotracer was performed using an integrated PET/MRI system (Siemens Biograph mMR; Siemens Healthcare). The PET/MRI scan was performed as a dynamic 60-min scan after injection of approximately 200 MBq (median : 202; range: 83-222 MBq) ⁶⁸Ga-NOTA-AE105.

If the patients were not eligible for a PET/MRI scan due to contraindications, a PET/CT scan was performed using a Biograph 128 mCT PET/CT device (Siemens Medical Solutions) with an axial field of view of 21.6 cm. However, of the 24 patients available for final analysis only 1 patient had undergone PET/CT instead of PET/MRI (see below).

PET images were reconstructed using a Deep Learning-based pseudoCT (22) attenuation map based on a UTE MRI sequence with absolute scatter correction (3-dimensional ordinary Poisson-ordered-subset expectation maximization [3D- OP-OSEM], 4 iterations, 21 subsets, 3.5mm gaussian filter). Static images were reconstructed using data acquired from 20-40 min, 40-60 min along with a dynamic 0-60 min series following injection of ⁶⁸Ga-NOTA-AE105. The reconstructed PET MRI images 20-40 min following tracer injection were used for further interpretation, quantification, and analysis.

MRI Protocol

The MRI scan protocol included a UTE AC sequence, a 3D Tl-weighted (T1W) MPRAGE both pre- and post-contrast injection with gadolinium, a T2-weighted (T2W) dark-fluid turbo inversion recovery magnitude (TIRM) (FLAIR) in both axial and coronal plane, a diffusion-weighted (DWI) RESOLVE, and a T2W BLADE. Parameters are listed in Table 1.

Table 1. MR parameters

Image Analysis

The analysis of the reconstructed image data was performed independently by a board-certified specialist in nuclear medicine and a board-certified specialist in neuroradiology. Each specialist was blinded to the clinical data. Tumors were delineated by drawing VOI's on the PET images and measured as maximum standardized uptake values (SUVmax). If no uPAR positive lesion was visible on the PET image, the MRI or CT image was used to delineate the tumors for SUVmax measurement. Reference brain VOI's running parallel to the cortex were drawn on the contralateral normal brain hemisphere at a single slice at the level of centrum semi-ovale. The VOI's were displaced approximately 7 mm from the cortical edge to avoid blood pool activity spill-in and mean standardized uptake value (SUVmean) was measured. A lesion was considered uPAR positive if TumorSUVmax-to-Normal-BrainSUVmean ratio (TBR) was at least 2.0 as used in a previous uPAR-PET study (17). Tumor size was measured on axial T2W FLAIR MRI images or axial CT images as the product of the maximal perpendicular diameters according to the Response Assessment in Neuro-Oncology (RANG) criteria (23). If no lesion was visible on the CT image, the previous MRI scan closest to the PET/CT scan was selected for tumor size measurement.

Follow Up

The patients were followed routinely at the Department of Oncology at Copenhagen University Hospital, Rigshospitalet. The follow up regime was standardized according to the national Danish glioma guidelines published by the Danish Neuro-Oncological Group (DNOG) (24). Final follow-up for endpoints was performed November 29, 2022. PFS was evaluated using the RANG criteria and defined as the time from uPAR-PET/MRI scan to progression (23,25). OS was defined as the time from uPAR-PET/MRI scan to the time of death. If there was no progression at the time of follow-up, the patient was censored according to the date of the most recent clinical follow-up visit.

Sample size was calculated based on disease prevalence and a follow-up period of 36 months. A total of 29 patients were required in order to detect significant differences in PFS and OS (risk of type I error of 0.05 and power of 0.8). Accounting for potential dropouts 30-35 patients were planned for enrollment in the trial. All continuous variables are reported as mean values with standard deviation (SD) or median with range. Kaplan-Meier analyses were performed for PFS and OS estimation, and inverse Kaplan-Meier for median follow-up time. Univariate Cox regression analysis was performed for OS and PFS with uPAR SUVmax as a continuous variable. To establish the optimal cutoff for uPAR SUVmax 20-40min after tracer injection, we used the Cutoff Finder application (26). P values less than 0.05 were considered statistically significant. R, version 4.2.2 (R Foundation for Statistical Computing) was used for data analysis.

Example 2 - Patients and image acquisition

A total of 33 patients were enrolled in the trial between March 2017 and June 2022. Out of these, 29 patients underwent imaging with a dynamic PET/MRI (n=26) or PET/CT (n=3) brain scan. Four patients were excluded due to failed radiopharmaceutical production (n=3) and technical issues (n = l). Data was available for reconstruction from 27 of these patients. Histology was available for all 27 patients and was reviewed in accordance with the 2021 WHO classification of central nervous system diseases (2). Three patients were excluded as they were diagnosed with central nervous system lymphoma. The final trial population thus constituted of 24 patients diagnosed with primary glioma where 23 patients underwent a PET/MRI scan and 1 patient underwent PET/CT scan, see Figure 1.

Demographic data from the 24 patients is summarized in Table 2.

Table 2. Baseline characteristics

The majority of the patients were diagnosed with WHO grade 4 gliomas (67%, 16/24), followed by grade 3 (25 %, 6/24), and grade 2 (8%, 2/24). Most tumors were located in the corpus callosum (21% (5/24), the frontal lobe (25%, 6/24) or the temporal lobe (21%, 5/24). No patients were worse than WHO performance status 1. The median tumor size was 1,700 mm² (range: 320-3,220 mm²). The median time from PET/MRI scan to surgery was 1 day (range, 0-21 days). The median injected dose of the tracer ⁶⁸Ga-NOTA-AE105 was 5.0 ml (range, 0.3ml- 7.5ml), and the median activity was 202 MBq (range, 83-222 MBq). No related adverse events or serious adverse events were recorded during the trial period.

Conclusion uPAR-PET imaging is feasible and safe in glioma patients.

Example 3 - Image analysis

Out of the 24 patients, 16 (67%, 16/24) were PET positive. Of the PET positive patients, 15 (94%, 15/16) had contrast enhancement on MRI, whereas one patient (6%, 1/16) had no MRI contrast enhancement (4 %, 1/24). Out of the 24 patients, 8 (33%, 8/24) were PET negative. Of the PET negative patients, 8 (100%, 8/8) had no pathological contrast enhancement. Lesions that were uPAR positive were seen primarily among the WHO grade 4 gliomas (94%, 15/16), with one WHO grade 3 glioma patient also presenting a PET positive tumor. Representative examples of PET positive tumor lesions are displayed in Figures 2 and 3. Conclusion

Thus, uPAR PET can identify high-grade gliomas (WHO grade 3 and 4) and demonstrate a high uptake and eligibility for uPAR-targeting radionuclide therapy.

Example 4 - Follow-up

The median follow-up time from uPAR-PET/MRI scan to PFS, OS or when the patients were censored was 9.8 (IQR, 7.2-26.4) months. A total of 16 (67%) patients experienced disease progression (15 grade 4 and 1 grade 2) and 10 (42%) patients died (all grade 4). First-line surgical and oncological treatment in the follow up period is illustrated in Table 2 above. All patients received surgical treatment, more than half of the patients underwent surgical resection (54%, 13/24), while the rest underwent biopsy (46%, 11/24). The most common oncological treatment was concomitant radio-, and chemotherapy (54%, 13/24), however some patients only received radiotherapy (38%, 9/24) or no adjuvant treatment (8%, 2/24).

Conclusion

Patients were followed for longer time and were typically pretreated with concomitant surgery, radiotherapy and chemotherapy

Example 5 - Progression-Free Survival and Overall Survival

Aim of study

To determine Progression-Free Survival (PFS) and Overall Survival (OS), based on uPAR imaging.

Results

Using the CutoffFinder program, optimal cutoff points for OS and PFS for the group all primary glioma (n = 24) by SUVmax were 1.1 for OS and 0.64 for PFS.

Using these cutoffs, uPAR expression was dichotomized into high and low and revealed a significantly worse prognosis in terms of OS and PFS with a HR of 10.5 (95% CI, 1.31-83.1; P=0.027), and HR of 17.3 (95% CI, 2.22-134.0; P=0.0064), respectively (Figures 4A and 4B). uPAR expression as a continuous variable was also associated with worse prognosis in terms of OS and PFS with HR of 2.48 (95% CI, 1.26-4.88;

P=0.0084), and HR of 2.28 (95% CI, 1.35-3.86; P=0.0020), respectively.

Additional subgroup analysis based on the primary HGGs only (n = 22) was also performed. Optimal cutoff point for OS and PFS for the high-grade group by SUVmax was 1.1 for both OS and PFS. uPAR expression dichotomized into high and low also for this subgroup showed significant worse prognosis for high compared to low uPAR uptake in terms of PFS with HR of 13.4 (95% CI, 1.70- 102.0; P=0.014) and borderline-significant worse prognosis with regard to OS with HR of 7.44 (95% CI, 0.94-59.0; P=0.058) (Figure 5A and 5B).

Furthermore, analysis of uPAR expression as a continuous variable for the subgroup HGG was also associated with worse prognosis in terms of both OS and PFS with HR of 2.23 (95% CI, 1.11-4.50; P=0.025), and HR of 2.11 (95% CI, 1.24-3.61; P=0.0063), respectively.

Conclusion

It was found that uPAR-PET was a strong prognostication tool.

Example 6 - Image analysis and comparison with somatostatin receptor imaging

Surprisingly there was not full concordance between contrast enhancement on MRI and high uptake on uPAR-PET demonstrating the value of uPAR-PET even when MRI is available. Furthermore, uPAR-PET was significant as a continuous variable regarding OS and PFS demonstrating a difference from contrast enhancement on MRI that is classified into either enhancement or not.

Discussion of data

In the current study, it was found that uPAR-PET activity measured as TumorSUVmax predicted a worse outcome with regard to OS and PFS for patients with primary gliomas. One may attribute this effect to the difference in survival expectancy between the LGG that were uPAR negative and the HGG that constituted the majority of our cohort. However, even when performing the analysis only for HGG uPAR PET was still prognostic. Consequently, uPAR-PET may be used for prognostication and treatment planning, e.g. surgical strategy in these patients. Additionally, we found the majority (67%) of the glioma patients to be uPAR-PET positive, which may be encouraging for further development of uPAR- PRRT for use in glioma patients.

Together, these findings highlight the potential of uPAR as a therapeutic target in gliomas and most importantly as a target for uPAR-PRRT. In particular, it should be noted that the positive uptake on uPAR-PET suggests that uPAR-PRRT using a similar ligand, but labelled with a therapeutic alpha or beta emitter, may be administered systemically rather than intratumorally.

It should be noted that external radiotherapy is well established in the treatment of HGG paving the way for targeted radioligand therapy in these patients. PRRT for brain tumors as a highly localized treatment modality is preferable to less precise external radiation therapy as it potentially may reduce the well-known cognitive side effects associated with external radiotherapy due to whole brain irradiation. Established radioligand therapies towards somatostatin receptors (SSTR-PRRT), primarily used for neuroendocrine neoplasms, have also been pursued in gliomas. The expression of SSTR has been reported in approximately 25% of gliomas with variable expression between LGG and HGG but with decreasing expression of SSTR2 in the most aggressive gliomas (27). In contrast, we found 94% of WHO grade 4 tumors to be uPAR-PET positive.

PRRT targeting SSTR was investigated in a study where 10 patients with WHO grade 2-3 gliomas were treated with intratumoral injections of ⁹⁰Y-DOTATOC. The ⁹⁰Y-DOTATOC treatment was reported to be both safe and effective in halting tumor progression for at least 13-45 months (28). Following this, another study demonstrated in a similar fashion the safety and efficacy of ⁹⁰Y-DOTATOC treatment of 3 patients with recurrent glioblastoma in 2010 (29). In recent years, there has been an increased focus on alpha-emitting PRRT targeting the neurokinin type 1 receptor (NK1R) (30). Interestingly, PRRT treatment with the alpha-emitting ²¹³Bi-DOTA-substance P with intratumoral administration has been demonstrated to be safe in 9 patients with recurrent glioblastomas (31). Thus, PRRT for gliomas is already under thorough investigation and so far, intratumoral alpha-emitting PRRT has been reported to be safe, feasible, and effective in facilitating clinically meaningful response in several clinical studies underlining the promising role of PRRT as an alternative to conventional therapies against gliomas. uPAR shows promise for targeted treatment in cancer due to its central role in tumor invasion and metastasis. One reason behind this is the conceptual advantage of targeting a receptor that is predominantly overexpressed in the most aggressive and actively invasive part of the tumors. The data from this study where we found that the majority of the patients displayed uPAR expression and that uPAR expression correlated with worsened outcome, is supported by existing literature where high expression of uPAR, especially in HGG, is found and correlated with poor prognosis (32). This emphasizes the role of uPAR as a desirable target expressed in the majority of HGG where therapy can be directed towards the most aggressive parts of the tumor. Several therapies targeting uPAR have or are currently undergoing investigation but have not been approved for clinical use 33,34}. Our group published a preclinical paper on uPAR-targeted PRRT with ¹⁷⁷Lu-DOTA-AE105 treatment of xenografts with colorectal cancer (20).

In this study, we showed a significant reduction of tumor size with good tolerability among the mice. Similarly, we demonstrated the efficacy of ¹⁷⁷Lu- DOTA-AE105 in treatment in a disseminated metastatic prostate cancer model (21). Accordingly, PRRT treatment targeting uPAR seems to have a great potential in several tumor types but is yet to be investigated in a clinical setting. An advantage of PRRT treatment with ¹⁷⁷Lu-DOTA-AE105 is that it is based on the same uPAR binding peptide, AE105, as ⁶⁸Ga-NOTA-AE105 implying the use of uPAR-PET as a companion diagnostic for treatment planning, monitoring, and dosimetry estimation in a uPAR-PRRT theragnostic approach in gliomas.

Although prolonged OS and PFS are the desired objectives of PRRT in patients with gliomas, replacing external radiotherapy may lower the side effects to healthy brain due to more specific tumor tissue targeting.

Conclusion

We demonstrate that uPAR expression as measured by uPAR-PET is significantly correlated with a worse outcome for patients with primary gliomas for both OS and PFS indicating the prognostic value of the uPAR radiotracer (exemplified by ⁶⁸Ga-NOTA-AE105). This emphasized uPAR as a promising target for diagnosis, prognostication, and targeted therapy against gliomas. Most importantly, uPAR holds great potential as a therapeutic target for PRRT treatment where uPAR-PET will serve as a companion diagnostic in a theranostic approach to preselect patients for uPAR-PRRT.

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Claims

1. A positron-emitting imaging agent for use in the prognostication of Progression-Free Survival (PFS) and/or Overall Survival (OS) of a brain tumour in a patient by PET imaging of the cancer, wherein said imaging agent comprises a uPAR binding peptide coupled to a radionuclide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; wherein the uPAR binding variant is selected from the group consisting of

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- (Tyr)-(Leu)([beta]-l-naphthyl-L-alanine)-(Ser), • (D-Glu)-([ beta] -cyclohexyl-L-a Ian ine)-(Phe)-(D-Ser)-(Tyr)- (Tyr)-(Leu)-(Trp)-(Ser),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3- dimethoxybenzyl)glycine)-(Ile); wherein a SUVmax and/or SUVmean level above a threshold level is indicative of a poor PFS prognosis and/or poor OS prognosis; and wherein a SUVmax and/or SUVmean level equal to or below a threshold level is indicative of good PFS prognosis and/or good OS prognosis.

2. The imaging agent for use according to claim 1, wherein the peptide is (D- Asp)-( [beta] -cyclohexyl-L-a la n ine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)- (Ser).

3. The imaging agent for use according to claim 1 or 2, wherein the radionuclide is selected from the group consisting of ⁶⁸Ga and ⁶⁴Cu.

4. The imaging agent for use according to any of the preceding claims, wherein the radionuclide is coupled to the uPAR binding peptide via a chelating agent, such as DOTA or NOTA.

5. The imaging agent for use according to any of the preceding claims, wherein the radionuclide is ⁶⁸Ga coupled to the uPAR binding peptide via a chelating agent NOTA.

6. The imaging agent for use according to any of the preceding claims, having the formula

7. The imaging agent for use according to any of the preceding claims 1-4, wherein the radionuclide is ⁶⁴Cu coupled to the uPAR binding peptide via a chelating agent DOTA or NOTA.

8. The imaging agent for use according to any of the preceding claims, wherein the imaging agent is to be administered in a dose of 10-500 MBq followed by PET scanning 10 min to 24 hours after the imaging agent has been administered, and quantification through SUVmax and/or SUVmean.

9. The imaging agent for use according to any of the preceding claims, wherein

- said SUVmax and/or SUVmean threshold in relation to PFS for all gliomas is in the range 0.3-1, preferably 0.4-0.8, more preferably around 0.64; and/or said SUVmax and/or SUVmean threshold in relation to OS for all gliomas is in the range 0.7-1.5, preferably 0.9-1.3, more preferably around 1.1.

10. The imaging agent for use according to any of the preceding claims 1-8, wherein

- said SUVmax and/or SUVmean threshold in relation to PFS for high-grade gliomas is in the range 0.7-1.5, preferably 0.9-1.3, more preferably around 1.1; and/or

- said SUVmax and/or SUVmean threshold in relation to OS for all high-grade gliomas is in the range 0.7-1.5, preferably 0.9-1.3, more preferably around 1.1.

11. The imaging agent for use according to any of the preceding claims, wherein the brain tumor is a high-grade glioma (WHO grade 3 and 4) or a low-grade glioma (WHO grade 1 and 2), preferably a high-grade glioma (WHO grade 4) (glioblastoma).

12. The imaging agent for use according to any of the preceding claims, wherein the brain tumor is a high-grade glioma (glioblastoma).

13. A positron-emitting imaging agent for use as companion diagnostic of a brain tumour patient by PET imaging of the cancer, wherein said imaging agent comprises a uPAR binding peptide coupled to a radionuclide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; wherein o a SUVmax and/or SUVmean level above a threshold level is indicative of that a uPAR binding drug, such as a uPAR binding radiopharmaceutical will be effective against said brain tumour; and o a SUVmax and/or SUVmean level equal to or below a threshold level is indicative of that a uPAR drug, such as a uPAR binding radiopharmaceutical will not be effective against said brain tumour; wherein the uPAR binding variant is selected from the group consisting of

14. The positron-emitting imaging agent for use according to claim 13, wherein the radionuclide and chelator are ⁶⁸Ga-NOTA.

15. The positron-emitting imaging agent for use according to claim 13, wherein the radionuclide and chelator are ⁶⁴Cu-DOTA.

16. The positron-emitting imaging agent for use according to any of claims 13-15, being a companion diagnostic for a radiopharmaceutical.

17. The positron-emitting imaging agent for use according to any of claims 13-16, being a companion diagnostic for a radiopharmaceutical as defined in any of claims 24-30.

18 The positron-emitting imaging agent for use according to claim 17, being a companion diagnostic for a radiopharmaceutical as defined in claim 24, wherein the radionuclide and chelator of the radiopharmaceutical is ¹⁷⁷Lu- DOTA.

19. The positron-emitting imaging agent for use according to any of claims 13-18, being a companion diagnostic for a radiopharmaceutical having the formula:

20. The positron-emitting imaging agent for use according to any of claims 13-19, wherein the companion diagnostic is for determining the applicability of a radiopharmaceutical to a specific person, such as a radiopharmaceutical as defined in any of claims 24-30.

21. The positron-emitting imaging agent for use according to any of claims 13-20, wherein the companion diagnostic is for determining the applicability of a radiopharmaceutical as defined in any of claims 24-30.

22. The positron-emitting imaging agent for use according to any of claims 13-21, wherein the brain tumor is a high-grade glioma (WHO grade 3 and 4) or a low-grade glioma (WHO grade 1 and 2), preferably a high-grade glioma (WHO grade 4) (glioblastoma).

23. The positron-emitting imaging agent for use according to any of claims 13-22, wherein the brain tumor is a high-grade glioma (glioblastoma).

24. A composition comprising a radiopharmaceutical for use in the treatment or alleviation of a brain tumor in a subject; wherein said radiopharmaceutical comprises a radionuclide and a uPAR binding peptide; and wherein the uPAR binding peptide is (D-Asp)-([beta]-cyclohexyl-L-alanine)- (Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser) or a uPAR binding variant thereof; and wherein the uPAR binding variant is selected from the group consisting of

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- (Leu)-(Leu)-(Trp)-(D-His), • (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)- ([beta]-cyclohexyl-L-alanine)-(Leu)-(Trp)-(Ile),

• (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N- (2,3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(2,3- dimethoxybenzyl)glycine)-(Ile); wherein said subject has been diagnosed with a uPAR expressing brain tumor and wherein said uPAR expression is above a predetermined threshold level, wherein said threshold level is determined using a positron-emitting imaging agent as defined in any of claims 1-12.

25. The composition for use according to claim 24, wherein 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, ²²⁷Th, preferably selected from ¹⁷⁷Lu, ⁶⁷Cu, ⁹⁰Y, ²¹¹At, ²²⁵Ac, and ²²⁷Th, and more preferably being ¹⁷⁷Lu.

26. The composition for use according to claim 24 or 25, wherein said radiopharmaceutical is coupled to the uPAR binding peptide via a chelator, such as the chelator being 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, H6phospa, PCTA, H2dedpa, H4octapa, H2azapa, H5decapa, HBED, HBED-cc, SHBED, BPCA, CP256, HEHA, PEPA and RESCA1, preferably selected from any of DOTA, NOTA, CB-TE2A, NODAGA, DFO, HBED, and HBED-cc, more preferably the chelator is DOTA or NOTA.

27. The composition for use according to any of claims 24-26, wherein the radionuclide and chelator is ¹⁷⁷Lu-DOTA.

28. The composition for use according to any of claims 24-27, wherein the radiopharmaceutical has the formula :

29. The positron-emitting imaging agent for use according to any of claims 24-28, wherein the brain tumor is a high-grade glioma (WHO grade 3 and 4) or a low-grade glioma (WHO grade 1 and 2), preferably a high-grade glioma (WHO grade 4) (glioblastoma).

30. The positron-emitting imaging agent for use according to any of claims 24-29, wherein the brain tumor is a high-grade glioma (glioblastoma).